Compositions and methods for the therapy and diagnosis of prostate cancer

ABSTRACT

Compositions and methods for the therapy and diagnosis of cancer, such as prostate cancer, are disclosed. Compositions may comprise one or more prostate-specific proteins, immunogenic portions thereof, or polynucleotides that encode such portions. Alternatively, a therapeutic composition may comprise an antigen presenting cell that expresses a prostate-specific protein, or a T cell that is specific for cells expressing such a protein. Such compositions may be used, for example, for the prevention and treatment of diseases such as prostate cancer. Diagnostic methods based on detecting a prostate-specific protein, or mRNA encoding such a protein, in a sample are also provided.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.09/605,783, filed Jun. 27, 2000, which is a continuation-in-part of U.S.patent application Ser. No. 09/593,793, filed Jun. 13, 2000, which is acontinuation-in-part of U.S. patent application Ser. No. 09/570,737,filed May 12, 2000, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/568,100, filed May 9, 2000, which is acontinuation-in-part of U.S. patent application Ser. No. 09/536,857,filed Mar. 27, 2000, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/483,672, filed Jan. 14, 2000, which is acontinuation-in-part of U.S. patent application Ser. No. 09/443,686,filed Nov. 18, 1999, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/439,313, filed Nov. 12, 1999, which is acontinuation-in-part of U.S. patent application Ser. No. 09/352,616,filed Jul. 13, 1999, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/288,946, filed Apr. 9, 1999, which is acontinuation-in-part of U.S. patent application No. 09/232,149, filedJan. 15, 1999, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/159,812, filed Sep. 23, 1998, which is acontinuation-in-part of U.S. patent application Ser. No. 09/115,453,filed Jul. 14, 1998, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/030,607, filed Feb. 25, 1998, which is acontinuation-in-part of U.S. patent application Ser. No. 09/020,956,filed Feb. 9, 1998.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to therapy and diagnosis ofcancer, such as prostate cancer. The invention is more specificallyrelated to polypeptides comprising at least a portion of aprostate-specific protein, and to polynucleotides encoding suchpolypeptides. Such polypeptides and polynucleotides may be used incompositions for prevention and treatment of prostate cancer, and forthe diagnosis and monitoring of such cancers.

BACKGROUND OF THE INVENTION

Cancer is a significant health problem throughout the world. AlthoughCancer is a significant health problem throughout the world. Althoughadvances have been made in detection and therapy of cancer, no vaccineor other universally successful method for prevention or treatment iscurrently available. Current therapies, which are generally based on acombination of chemotherapy or surgery and radiation, continue to proveinadequate in many patients.

Prostate cancer is the most common form of cancer among males, with anestimated incidence of 30% in men over the age of 50. Overwhelmingclinical evidence shows that human prostate cancer has the propensity tometastasize to bone, and the disease appears to progress inevitably fromandrogen dependent to androgen refractory status, leading to increasedpatient mortality. This prevalent disease is currently the secondleading cause of cancer death among men in the U.S.

In spite of considerable research into therapies for the disease,prostate cancer remains difficult to treat. Commonly, treatment is basedon surgery and/or radiation therapy, but these methods are ineffectivein a significant percentage of cases. Two previously identified prostatespecific proteins—prostate specific antigen (PSA) and prostatic acidphosphatase (PAP)—have limited therapeutic and diagnostic potential. Forexample, PSA levels do not always correlate well with the presence ofprostate cancer, being positive in a percentage of non-prostate cancercases, including benign prostatic hyperplasia (BPH). Furthermore, PSAmeasurements correlate with prostate volume, and do not indicate thelevel of metastasis.

In spite of considerable research into therapies for these and othercancers, prostate cancer remains difficult to diagnose and treateffectively. Accordingly, there is a need in the art for improvedmethods for detecting and treating such cancers. The present inventionfulfills these needs and further provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methodsfor the diagnosis and therapy of cancer, such as prostate cancer. In oneaspect, the present invention provides polypeptides comprising at leasta portion of a prostate-specific protein, or a variant thereof. Certainportions and other variants are immunogenic, such that the ability ofthe variant to react with antigen-specific antisera is not substantiallydiminished. Within certain embodiments, the polypeptide comprises asequence that is encoded by a polynucleotide sequence selected from thegroup consisting of: (a) sequences recited in SEQ ID NO: 1-111, 115-171,173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381,382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587,591, 593-606, 618-705, 709-774, 777, 789, 817, 823 and 824; (b) variantsof a sequence recited in SEQ ID NO: 1-111, 115-171, 173-175, 177,179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476,524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606,618-705, 709-774, 777, 789, 817, 823 and 824; and (c) complements of asequence of (a) or (b). In specific embodiments, the polypeptides of thepresent invention comprise at least a portion of a tumor protein thatincludes an amino acid sequence selected from the group consisting ofsequences recited in SEQ ID NO: 112-114, 172, 176, 178, 327, 329, 331,336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527,532, 534, 537-551, 553-568, 573-586, 588-590, 592, 706-708, 775, 776,778, 780, 781, 811, 814, 818, 826 and 827, and variants thereof.

The present invention further provides polynucleotides that encode apolypeptide as described above, or a portion thereof (such as a portionencoding at least 15 amino acid residues of a prostate-specificprotein), expression vectors comprising such polynucleotides and hostcells transformed or transfected with such expression vectors.

Within other aspects, the present invention provides pharmaceuticalcompositions comprising a polypeptide or polynucleotide as describedabove and a physiologically acceptable carrier.

Within a related aspect of the present invention, immunogeniccompositions, or vaccines for prophylactic or therapeutic use areprovided. Such compositions comprise a polypeptide or polynucleotide asdescribed above and an immunostimulant.

The present invention further provides pharmaceutical compositions thatcomprise: (a) an antibody or antigen-binding fragment thereof thatspecifically binds to a prostate-specific protein; and (b) aphysiologically acceptable carrier.

Within further aspects, the present invention provides pharmaceuticalcompositions comprising: (a) an antigen presenting cell that expresses apolypeptide as described above and (b) a pharmaceutically acceptablecarrier or excipient. Antigen presenting cells include dendritic cells,macrophages, monocytes, fibroblasts and B cells.

Within related aspects, immunogenic compositions, or vaccines, areprovided that comprise: (a) an antigen presenting cell that expresses apolypeptide as described above and (b) an immunostimulant.

The present invention further provides, in other aspects, fusionproteins that comprise at least one polypeptide as described above, aswell as polynucleotides encoding such fusion proteins.

Within related aspects, pharmaceutical compositions comprising a fusionprotein, or a polynucleotide encoding a fusion protein, in combinationwith a physiologically acceptable carrier are provided.

Compositions are further provided, within other aspects, that comprise afusion protein, or a polynucleotide encoding a fusion protein, incombination with an immunostimulant.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient a composition as recited above. The patientmay be afflicted with prostate cancer, in which case the methods providetreatment for the disease, or patient considered at risk for such adisease may be treated prophylactically.

The present invention further provides, within other aspects, methodsfor removing tumor cells from a biological sample, comprising contactinga biological sample with T cells that specifically react with aprostate-specific protein, wherein the step of contacting is performedunder conditions and for a time sufficient to permit the removal ofcells expressing the protein from the sample.

Within related aspects, methods are provided for inhibiting thedevelopment of a cancer in a patient, comprising administering to apatient a biological sample treated as described above.

Methods are further provided, within other aspects, for stimulatingand/or expanding T cells specific for a prostate-specific protein,comprising contacting T cells with one or more of: (i) a polypeptide asdescribed above; (ii) a polynucleotide encoding such a polypeptide;and/or (iii) an antigen presenting cell that expresses such apolypeptide; under conditions and for a time sufficient to permit thestimulation and/or expansion of T cells. Isolated T cell populationscomprising T cells prepared as described above are also provided.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient an effective amount of a T cell population asdescribed above.

The present invention further provides methods for inhibiting thedevelopment of a cancer in a patient, comprising the steps of: (a)incubating CD4⁺ and/or CD8⁺ T cells isolated from a patient with one ormore of: (i) a polypeptide comprising at least an immunogenic portion ofa prostate-specific protein; (ii) a polynucleotide encoding such apolypeptide; and (iii) an antigen-presenting cell that expressed such apolypeptide; and (b) administering to the patient an effective amount ofthe proliferated T cells, and thereby inhibiting the development of acancer in the patient. Proliferated cells may, but need not, be clonedprior to administration to the patient.

Within further aspects, the present invention provides methods fordetermining the presence or absence of a cancer in a patient,comprising: (a) contacting a biological sample obtained from a patientwith a binding agent that binds to a polypeptide as recited above; (b)detecting in the sample an amount of polypeptide that binds to thebinding agent; and (c) comparing the amount of polypeptide with apredetermined cut-off value, and therefrom determining the presence orabsence of a cancer in the patient. Within preferred embodiments, thebinding agent is an antibody, more preferably a monoclonal antibody. Thecancer may be prostate cancer.

The present invention also provides, within other aspects, methods formonitoring the progression of a cancer in a patient. Such methodscomprise the steps of: (a) contacting a biological sample obtained froma patient at a first point in time with a binding agent that binds to apolypeptide as recited above; (b) detecting in the sample an amount ofpolypeptide that binds to the binding agent; (c) repeating steps (a) and(b) using a biological sample obtained from the patient at a subsequentpoint in time; and (d) comparing the amount of polypeptide detected instep (c) with the amount detected in step (b) and therefrom monitoringthe progression of the cancer in the patient.

The present invention further provides, within other aspects, methodsfor determining the presence or absence of a cancer in a patient,comprising the steps of: (a) contacting a biological sample obtainedfrom a patient with an oligonucleotide that hybridizes to apolynucleotide that encodes a prostate-specific protein; (b) detectingin the sample a level of a polynucleotide, preferably mRNA, thathybridizes to the oligonucleotide; and (c) comparing the level ofpolynucleotide that hybridizes to the oligonucleotide with apredetermined cut-off value, and therefrom determining the presence orabsence of a cancer in the patient. Within certain embodiments, theamount of mRNA is detected via polymerase chain reaction using, forexample, at least one oligonucleotide primer that hybridizes to apolynucleotide encoding a polypeptide as recited above, or a complementof such a polynucleotide. Within other embodiments, the amount of mRNAis detected using a hybridization technique, employing anoligonucleotide probe that hybridizes to a polynucleotide that encodes apolypeptide as recited above, or a complement of such a polynucleotide.

In related aspects, methods are provided for monitoring the progressionof a cancer in a patient, comprising the steps of: (a) contacting abiological sample obtained from a patient with an oligonucleotide thathybridizes to a polynucleotide that encodes a prostate-specific protein;(b) detecting in the sample an amount of a polynucleotide thathybridizes to the oligonucleotide; (c) repeating steps (a) and (b) usinga biological sample obtained from the patient at a subsequent point intime; and (d) comparing the amount of polynucleotide detected in step(c) with the amount detected in step (b) and therefrom monitoring theprogression of the cancer in the patient.

Within further aspects, the present invention provides antibodies, suchas monoclonal antibodies, that bind to a polypeptide as described above,as well as diagnostic kits comprising such antibodies. Diagnostic kitscomprising one or more oligonucleotide probes or primers as describedabove are also provided.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE IDENTIFIERS

FIG. 1 illustrates the ability of T cells to kill fibroblasts expressingthe representative prostate-specific polypeptide P502S, as compared tocontrol fibroblasts. The percentage lysis is shown as a series ofeffector:target ratios, as indicated.

FIGS. 2A and 2B illustrate the ability of T cells to recognize cellsexpressing the representative prostate-specific polypeptide P502S. Ineach case, the number of γ-interferon spots is shown for differentnumbers of responders. In FIG. 2A, data is presented for fibroblastspulsed with the P2S-12 peptide, as compared to fibroblasts pulsed with acontrol E75 peptide. In FIG. 2B, data is presented for fibroblastsexpressing P502S, as compared to fibroblasts expressing HER-2/neu.

FIG. 3 represents a peptide competition binding assay showing that theP1S#10 peptide, derived from P501S, binds HLA-A2. Peptide P1S#10inhibits HLA-A2 restricted presentation of fluM58 peptide to CTL cloneD150M58 in TNF release bioassay. D150M58 CTL is specific for the HLA-A2binding influenza matrix peptide fluM58.

FIG. 4 illustrates the ability of T cell lines generated from P1S#10immunized mice to specifically lyse P1S#10-pulsed Jurkat A2Kb targetsand P501S-transduced Jurkat A2Kb targets, as compared to EGFP-transducedJurkat A2Kb. The percent lysis is shown as a series of effector totarget ratios, as indicated.

FIG. 5 illustrates the ability of a T cell clone to recognize andspecifically lyse Jurkat A2Kb cells expressing the representativeprostate-specific polypeptide P501S, thereby demonstrating that theP1S#10 peptide may be a naturally processed epitope of the P501Spolypeptide.

FIGS. 6A and 6B are graphs illustrating the specificity of a CD8⁺ cellline (3A-1) for a representative prostate-specific antigen (P501S). FIG.6A shows the results of a ⁵¹Cr release assay. The percent specific lysisis shown as a series of effector:target ratios, as indicated. FIG. 6Bshows the production of interferon-gamma by 3A-1 cells stimulated withautologous B-LCL transduced with P501S, at varying effector:targetrations as indicated.

FIG. 7 is a Western blot showing the expression of P501S in baculovirus.

FIG. 8 illustrates the results of epitope mapping studies on P501S.

FIG. 9 is a schematic representation of the P501S protein showing thelocation of transmembrane domains and predicted intracellular andextracellular domains.

FIG. 10 is a genomic map showing the location of the prostate genesP775P, P704P, B305D, P712P and P774P within the Cat Eye Syndrome regionof chromosome 22q11.2

FIG. 11 shows the results of an ELISA assay to determine the specificityof rabbit polyclonal antisera raised against P501S.

FIGS. 12A(1), 12A(2), 12A(3), and B are the full-length cDNA (SEQ IDNO:591) and predicted amino acid (SEQ ID NO:592) sequences,respectively, for the clone P788P.

SEQ ID NO: 1 is the determined cDNA sequence for F1-13

SEQ ID NO: 2 is the determined 3′ cDNA sequence for F1-12

SEQ ID NO: 3 is the determined 5′ cDNA sequence for F1-12

SEQ ID NO: 4 is the determined 3′ cDNA sequence for F1-16

SEQ ID NO: 5 is the determined 3′ cDNA sequence for H1-1

SEQ ID NO: 6 is the determined 3′ cDNA sequence for H1-9

SEQ ID NO: 7 is the determined 3′ cDNA sequence for H1-4

SEQ ID NO: 8 is the determined 3′ cDNA sequence for J1-17

SEQ ID NO: 9 is the determined 5′ cDNA sequence for J1-17

SEQ ID NO: 10 is the determined 3′ cDNA sequence for L1-12

SEQ ID NO: 11 is the determined 5′ cDNA sequence for L1-12

SEQ ID NO: 12 is the determined 3′ cDNA sequence for N1-1862

SEQ ID NO: 13 is the determined 5′ cDNA sequence for N1-1862

SEQ ID NO: 14 is the determined 3′ cDNA sequence for J1-13

SEQ ID NO: 15 is the determined 5′ cDNA sequence for J1-13

SEQ ID NO: 16 is the determined 3′ cDNA sequence for J1-19

SEQ ID NO: 17 is the determined 5′ cDNA sequence for J1-19

SEQ ID NO: 18 is the determined 3′ cDNA sequence for J1-25

SEQ ID NO: 19 is the determined 5′ cDNA sequence for J1-25

SEQ ID NO: 20 is the determined 5′ cDNA sequence for J1-24

SEQ ID NO: 21 is the determined 3′ cDNA sequence for J1-24

SEQ ID NO: 22 is the determined 5′ cDNA sequence for K1-58

SEQ ID NO: 23 is the determined 3′ cDNA sequence for K1-58

SEQ ID NO: 24 is the determined 5′ cDNA sequence for K1-63

SEQ ID NO: 25 is the determined 3′ cDNA sequence for K1-63

SEQ ID NO: 26 is the determined 5′ cDNA sequence for L1-4

SEQ ID NO: 27 is the determined 3′ cDNA sequence for L1-4

SEQ ID NO: 28 is the determined 5′ cDNA sequence for L1-14

SEQ ID NO: 29 is the determined 3′ cDNA sequence for L1-14

SEQ ID NO: 30 is the determined 3′ cDNA sequence for J1-12

SEQ ID NO: 31 is the determined 3′ cDNA sequence for J1-16

SEQ ID NO: 32 is the determined 3′ cDNA sequence for J1-21

SEQ ID NO: 33 is the determined 3′ cDNA sequence for K1-48

SEQ ID NO: 34 is the determined 3′ cDNA sequence for K1-55

SEQ ID NO: 35 is the determined 3′ cDNA sequence for L1-2

SEQ ID NO: 36 is the determined 3′ cDNA sequence for L1-6

SEQ ID NO: 37 is the determined 3′ cDNA sequence for N1-1858

SEQ ID NO: 38 is the determined 3′ cDNA sequence for N1-1860

SEQ ID NO: 39 is the determined 3′ cDNA sequence for N1-1861

SEQ ID NO: 40 is the determined 3′ cDNA sequence for N1-1864

SEQ ID NO: 41 is the determined cDNA sequence for P5

SEQ ID NO: 42 is the determined cDNA sequence for P8

SEQ ID NO: 43 is the determined cDNA sequence for P9

SEQ ID NO: 44 is the determined cDNA sequence for P18

SEQ ID NO: 45 is the determined cDNA sequence for P20

SEQ ID NO: 46 is the determined cDNA sequence for P29

SEQ ID NO: 47 is the determined cDNA sequence for P30

SEQ ID NO: 48 is the determined cDNA sequence for P34

SEQ ID NO: 49 is the determined cDNA sequence for P36

SEQ ID NO: 50 is the determined cDNA sequence for P38

SEQ ID NO: 51 is the determined cDNA sequence for P39

SEQ ID NO: 52 is the determined cDNA sequence for P42

SEQ ID NO: 53 is the determined cDNA sequence for P47

SEQ ID NO: 54 is the determined cDNA sequence for P49

SEQ ID NO: 55 is the determined cDNA sequence for P50

SEQ ID NO: 56 is the determined cDNA sequence for P53

SEQ ID NO: 57 is the determined cDNA sequence for P55

SEQ ID NO: 58 is the determined cDNA sequence for P60

SEQ ID NO: 59 is the determined cDNA sequence for P64

SEQ ID NO: 60 is the determined cDNA sequence for P65

SEQ ID NO: 61 is the determined cDNA sequence for P73

SEQ ID NO: 62 is the determined cDNA sequence for P75

SEQ ID NO: 63 is the determined cDNA sequence for P76

SEQ ID NO: 64 is the determined cDNA sequence for P79

SEQ ID NO: 65 is the determined cDNA sequence for P84

SEQ ID NO: 66 is the determined cDNA sequence for P68

SEQ ID NO: 67 is the determined cDNA sequence for P80 (also referred toas P704P)

SEQ ID NO: 68 is the determined cDNA sequence for P82

SEQ ID NO: 69 is the determined cDNA sequence for U1-3064

SEQ ID NO: 70 is the determined cDNA sequence for U1-3065

SEQ ID NO: 71 is the determined cDNA sequence for V1-3692

SEQ ID NO: 72 is the determined cDNA sequence for 1A-3905

SEQ ID NO: 73 is the determined cDNA sequence for V1-3686

SEQ ID NO: 74 is the determined cDNA sequence for R1-2330

SEQ ID NO: 75 is the determined cDNA sequence for 1B-3976

SEQ ID NO: 76 is the determined cDNA sequence for V1-3679

SEQ ID NO: 77 is the determined cDNA sequence for 1G-4736

SEQ ID NO: 78 is the determined cDNA sequence for 1G-4738

SEQ ID NO: 79 is the determined cDNA sequence for 1G-4741

SEQ ID NO: 80 is the determined cDNA sequence for 1G-4744

SEQ ID NO: 81 is the determined cDNA sequence for 1G-4734

SEQ ID NO: 82 is the determined cDNA sequence for 1H-4774

SEQ ID NO: 83 is the determined cDNA sequence for 1H-4781

SEQ ID NO: 84 is the determined cDNA sequence for 1H-4785

SEQ ID NO: 85 is the determined cDNA sequence for 1H-4787

SEQ ID NO: 86 is the determined cDNA sequence for 1H-4796

SEQ ID NO: 87 is the determined cDNA sequence for 1H-4807

SEQ ID NO: 88 is the determined cDNA sequence for 1I-4810

SEQ ID NO: 89 is the determined cDNA sequence for 1I-4811

SEQ ID NO: 90 is the determined cDNA sequence for 1J-4876

SEQ ID NO: 91 is the determined cDNA sequence for 1K-4884

SEQ ID NO: 92 is the determined cDNA sequence for 1K-4896

SEQ ID NO: 93 is the determined cDNA sequence for 1G-4761

SEQ ID NO: 94 is the determined cDNA sequence for 1G-4762

SEQ ID NO: 95 is the determined cDNA sequence for 1H-4766

SEQ ID NO: 96 is the determined cDNA sequence for 1H-4770

SEQ ID NO: 97 is the determined cDNA sequence for 1H-4771

SEQ ID NO: 98 is the determined cDNA sequence for 1H-4772

SEQ ID NO: 99 is the determined cDNA sequence for 1D-4297

SEQ ID NO: 100 is the determined cDNA sequence for 1D-4309

SEQ ID NO: 101 is the determined cDNA sequence for 1D-4283

SEQ ID NO: 102 is the determined cDNA sequence for 1D-4288

SEQ ID NO: 103 is the determined cDNA sequence for 1D-4283

SEQ ID NO: 104 is the determined cDNA sequence for 1D-4304

SEQ ID NO: 105 is the determined cDNA sequence for 1D-4296

SEQ ID NO: 106 is the determined cDNA sequence for 1D-4280

SEQ ID NO: 107 is the determined full length cDNA sequence for F1-12(also referred to as P504S)

SEQ ID NO: 108 is the predicted amino acid sequence for F1-12

SEQ ID NO: 109 is the determined full length cDNA sequence for J1-12

SEQ ID NO: 110 is the determined full length cDNA sequence for L1-12(also referred to as P501S)

SEQ ID NO: 111 is the determined full length cDNA sequence for N1-1862also referred to as P503S)

SEQ ID NO: 112 is the predicted amino acid sequence for J1-17

SEQ ID NO: 113 is the predicted amino acid sequence for L1-12 (alsoreferred to as P501S)

SEQ ID NO: 114 is the predicted amino acid sequence for N1-1862 (alsoreferred to as P503S)

SEQ ID NO: 115 is the determined cDNA sequence for P89

SEQ ID NO: 116 is the determined cDNA sequence for P90

SEQ ID NO: 117 is the determined cDNA sequence for P92

SEQ ID NO: 118 is the determined cDNA sequence for P95

SEQ ID NO: 119 is the determined cDNA sequence for P98

SEQ ID NO: 120 is the determined cDNA sequence for P102

SEQ ID NO: 121 is the determined cDNA sequence for P110

SEQ ID NO: 122 is the determined cDNA sequence for P111

SEQ ID NO: 123 is the determined cDNA sequence for P114

SEQ ID NO: 124 is the determined cDNA sequence for P115

SEQ ID NO: 125 is the determined cDNA sequence for P116

SEQ ID NO: 126 is the determined cDNA sequence for P124

SEQ ID NO: 127 is the determined cDNA sequence for P126

SEQ ID NO: 128 is the determined cDNA sequence for P130

SEQ ID NO: 129 is the determined cDNA sequence for P133

SEQ ID NO: 130 is the determined cDNA sequence for P138

SEQ ID NO: 131 is the determined cDNA sequence for P143

SEQ ID NO: 132 is the determined cDNA sequence for P151

SEQ ID NO: 133 is the determined cDNA sequence for P156

SEQ ID NO: 134 is the determined cDNA sequence for P157

SEQ ID NO: 135 is the determined cDNA sequence for P166

SEQ ID NO: 136 is the determined cDNA sequence for P176

SEQ ID NO: 137 is the determined cDNA sequence for P178

SEQ ID NO: 138 is the determined cDNA sequence for P179

SEQ ID NO: 139 is the determined cDNA sequence for P185

SEQ ID NO: 140 is the determined cDNA sequence for P192

SEQ ID NO: 141 is the determined cDNA sequence for P201

SEQ ID NO: 142 is the determined cDNA sequence for P204

SEQ ID NO: 143 is the determined cDNA sequence for P208

SEQ ID NO: 144 is the determined cDNA sequence for P211

SEQ ID NO: 145 is the determined cDNA sequence for P213

SEQ ID NO: 146 is the determined cDNA sequence for P219

SEQ ID NO: 147 is the determined cDNA sequence for P237

SEQ ID NO: 148 is the determined cDNA sequence for P239

SEQ ID NO: 149 is the determined cDNA sequence for P248

SEQ ID NO: 150 is the determined cDNA sequence for P251

SEQ ID NO: 151 is the determined cDNA sequence for P255

SEQ ID NO: 152 is the determined cDNA sequence for P256

SEQ ID NO: 153 is the determined cDNA sequence for P259

SEQ ID NO: 154 is the determined cDNA sequence for P260

SEQ ID NO: 155 is the determined cDNA sequence for P263

SEQ ID NO: 156 is the determined cDNA sequence for P264

SEQ ID NO: 157 is the determined cDNA sequence for P266

SEQ ID NO: 158 is the determined cDNA sequence for P270

SEQ ID NO: 159 is the determined cDNA sequence for P272

SEQ ID NO: 160 is the determined cDNA sequence for P278

SEQ ID NO: 161 is the determined cDNA sequence for P105

SEQ ID NO: 162 is the determined cDNA sequence for P107

SEQ ID NO: 163 is the determined cDNA sequence for P137

SEQ ID NO: 164 is the determined cDNA sequence for P194

SEQ ID NO: 165 is the determined cDNA sequence for P195

SEQ ID NO: 166 is the determined cDNA sequence for P196

SEQ ID NO: 167 is the determined cDNA sequence for P220

SEQ ID NO: 168 is the determined cDNA sequence for P234

SEQ ID NO: 169 is the determined cDNA sequence for P235

SEQ ID NO: 170 is the determined cDNA sequence for P243

SEQ ID NO: 171 is the determined cDNA sequence for P703P-DE1

SEQ ID NO: 172 is the predicted amino acid sequence for P703P-DE1

SEQ ID NO: 173 is the determined cDNA sequence for P703P-DE2

SEQ ID NO: 174 is the determined cDNA sequence for P703P-DE6

SEQ ID NO: 175 is the determined cDNA sequence for P703P-DE13

SEQ ID NO: 176 is the predicted amino acid sequence for P703P-DE13

SEQ ID NO: 177 is the determined cDNA sequence for P703P-DE14

SEQ ID NO: 178 is the predicted amino acid sequence for P703P-DE14

SEQ ID NO: 179 is the determined extended cDNA sequence for 1G-4736

SEQ ID NO: 180 is the determined extended cDNA sequence for 1G-4738

SEQ ID NO: 181 is the determined extended cDNA sequence for 1G-4741

SEQ ID NO: 182 is the determined extended cDNA sequence for 1G-4744

SEQ ID NO: 183 is the determined extended cDNA sequence for 1H-4774

SEQ ID NO: 184 is the determined extended cDNA sequence for 1H-4781

SEQ ID NO: 185 is the determined extended cDNA sequence for 1H-4785

SEQ ID NO: 186 is the determined extended cDNA sequence for 1H-4787

SEQ ID NO: 187 is the determined extended cDNA sequence for 1H-4796

SEQ ID NO: 188 is the determined extended cDNA sequence for 1I-4807

SEQ ID NO: 189 is the determined 3′ cDNA sequence for 1I-4810

SEQ ID NO: 190 is the determined 3′ cDNA sequence for 1I-4811

SEQ ID NO: 191 is the determined extended cDNA sequence for 1J-4876

SEQ ID NO: 192 is the determined extended cDNA sequence for 1K-4884

SEQ ID NO: 193 is the determined extended cDNA sequence for 1K-4896

SEQ ID NO: 194 is the determined extended cDNA sequence for 1G-4761

SEQ ID NO: 195 is the determined extended cDNA sequence for 1G-4762

SEQ ID NO: 196 is the determined extended cDNA sequence for 1H-4766

SEQ ID NO: 197 is the determined 3′ cDNA sequence for 1H-4770

SEQ ID NO: 198 is the determined 3′ cDNA sequence for 1H-4771

SEQ ID NO: 199 is the determined extended cDNA sequence for 1H-4772

SEQ ID NO: 200 is the determined extended cDNA sequence for 1D-4309

SEQ ID NO: 201 is the determined extended cDNA sequence for 1D-4278

SEQ ID NO: 202 is the determined extended cDNA sequence for 1D-4288

SEQ ID NO: 203 is the determined extended cDNA sequence for 1D-4283

SEQ ID NO: 204 is the determined extended cDNA sequence for 1D-4304

SEQ ID NO: 205 is the determined extended cDNA sequence for 1D-4296

SEQ ID NO: 206 is the determined extended cDNA sequence for 1D-4280

SEQ ID NO: 207 is the determined cDNA sequence for 10-d8fwd

SEQ ID NO: 208 is the determined cDNA sequence for 10-H10con

SEQ ID NO: 209 is the determined cDNA sequence for 11-C8rev

SEQ ID NO: 210 is the determined cDNA sequence for 7.g6fwd

SEQ ID NO: 211 is the determined cDNA sequence for 7.g6rev

SEQ ID NO: 212 is the determined cDNA sequence for 8-b5fwd

SEQ ID NO: 213 is the determined cDNA sequence for 8-b5rev

SEQ ID NO: 214 is the determined cDNA sequence for 8-b6fwd

SEQ ID NO: 215 is the determined cDNA sequence for 8-d6rev

SEQ ID NO: 216 is the determined cDNA sequence for 8-d4fwd

SEQ ID NO: 217 is the determined cDNA sequence for 8-d9rev

SEQ ID NO: 218 is the determined cDNA sequence for 8-g3fwd

SEQ ID NO: 219 is the determined cDNA sequence for 8-g3rev

SEQ ID NO: 220 is the determined cDNA sequence for 8-h11rev

SEQ ID NO: 221 is the determined cDNA sequence for g-f12fwd

SEQ ID NO: 222 is the determined cDNA sequence for g-f3rev

SEQ ID NO: 223 is the determined cDNA sequence for P509S

SEQ ID NO: 224 is the determined cDNA sequence for P510S

SEQ ID NO: 225 is the determined cDNA sequence for P703DE5

SEQ ID NO: 226 is the determined cDNA sequence for 9-A11

SEQ ID NO: 227 is the determined cDNA sequence for 8-C6

SEQ ID NO: 228 is the determined cDNA sequence for 8-H7

SEQ ID NO: 229 is the determined cDNA sequence for JPTPN13

SEQ ID NO: 230 is the determined cDNA sequence for JPTPN14

SEQ ID NO: 231 is the determined cDNA sequence for JPTPN23

SEQ ID NO: 232 is the determined cDNA sequence for JPTPN24

SEQ ID NO: 233 is the determined cDNA sequence for JPTPN25

SEQ ID NO: 234 is the determined cDNA sequence for JPTPN30

SEQ ID NO: 235 is the determined cDNA sequence for JPTPN34

SEQ ID NO: 236 is the determined cDNA sequence for PTPN35

SEQ ID NO: 237 is the determined cDNA sequence for JPTPN36

SEQ ID NO: 238 is the determined cDNA sequence for JPTPN38

SEQ ID NO: 239 is the determined cDNA sequence for JPTPN39

SEQ ID NO: 240 is the determined cDNA sequence for JPTPN40

SEQ ID NO: 241 is the determined cDNA sequence for JPTPN41

SEQ ID NO: 242 is the determined cDNA sequence for JPTPN42

SEQ ID NO: 243 is the determined cDNA sequence for JPTPN45

SEQ ID NO: 244 is the determined cDNA sequence for JPTPN46

SEQ ID NO: 245 is the determined cDNA sequence for JPTPN51

SEQ ID NO: 246 is the determined cDNA sequence for JPTPN56

SEQ ID NO: 247 is the determined cDNA sequence for PTPN64

SEQ ID NO: 248 is the determined cDNA sequence for JPTPN65

SEQ ID NO: 249 is the determined cDNA sequence for JPTPN67

SEQ ID NO: 250 is the determined cDNA sequence for JPTPN76

SEQ ID NO: 251 is the determined cDNA sequence for JPTPN84

SEQ ID NO: 252 is the determined cDNA sequence for JPTPN85

SEQ ID NO: 253 is the determined cDNA sequence for JPTPN86

SEQ ID NO: 254 is the determined cDNA sequence for JPTPN87

SEQ ID NO: 255 is the determined cDNA sequence for JPTPN88

SEQ ID NO: 256 is the determined cDNA sequence for JP1F1

SEQ ID NO: 257 is the determined cDNA sequence for JP1F2

SEQ ID NO: 258 is the determined cDNA sequence for JP1C2

SEQ ID NO: 259 is the determined cDNA sequence for JP1B1

SEQ ID NO: 260 is the determined cDNA sequence for JP1B2

SEQ ID NO: 261 is the determined cDNA sequence for JP1D3

SEQ ID NO: 262 is the determined cDNA sequence for JP1A4

SEQ ID NO: 263 is the determined cDNA sequence for JP1F5

SEQ ID NO: 264 is the determined cDNA sequence for JP1E6

SEQ ID NO: 265 is the determined cDNA sequence for JP1D6

SEQ ID NO: 266 is the determined cDNA sequence for JP1B5

SEQ ID NO: 267 is the determined cDNA sequence for JP1A6

SEQ ID NO: 268 is the determined cDNA sequence for JP1E8

SEQ ID NO: 269 is the determined cDNA sequence for JP1D7

SEQ ID NO: 270 is the determined cDNA sequence for JP1D9

SEQ ID NO: 271 is the determined cDNA sequence for JP1C10

SEQ ID NO: 272 is the determined cDNA sequence for JP1A9

SEQ ID NO: 273 is the determined cDNA sequence for JP1F12

SEQ ID NO: 274 is the determined cDNA sequence for JP1E12

SEQ ID NO: 275 is the determined cDNA sequence for JP1D11

SEQ ID NO: 276 is the determined cDNA sequence for JP1C11

SEQ ID NO: 277 is the determined cDNA sequence for JP1C12

SEQ ID NO: 278 is the determined cDNA sequence for JP1B12

SEQ ID NO: 279 is the determined cDNA sequence for JP1A12

SEQ ID NO: 280 is the determined cDNA sequence for JP8G2

SEQ ID NO: 281 is the determined cDNA sequence for JP8H1

SEQ ID NO: 282 is the determined cDNA sequence for JP8H2

SEQ ID NO: 283 is the determined cDNA sequence for JP8A3

SEQ ID NO: 284 is the determined cDNA sequence for JP8A4

SEQ ID NO: 285 is the determined cDNA sequence for JP8C3

SEQ ID NO: 286 is the determined cDNA sequence for JP8G4

SEQ ID NO: 287 is the determined cDNA sequence for JP8B6

SEQ ID NO: 288 is the determined cDNA sequence for JP8D6

SEQ ID NO: 289 is the determined cDNA sequence for JP8F5

SEQ ID NO: 290 is the determined cDNA sequence for JP8A8

SEQ ID NO: 291 is the determined cDNA sequence for JP8C7

SEQ ID NO: 292 is the determined cDNA sequence for JP8D7

SEQ ID NO: 293 is the determined cDNA sequence for P8D8

SEQ ID NO: 294 is the determined cDNA sequence for JP8E7

SEQ ID NO: 295 is the determined cDNA sequence for JP8F8

SEQ ID NO: 296 is the determined cDNA sequence for JP8G8

SEQ ID NO: 297 is the determined cDNA sequence for JP8B10

SEQ ID NO: 298 is the determined cDNA sequence for JP8C10

SEQ ID NO: 299 is the determined cDNA sequence for JP8E9

SEQ ID NO: 300 is the determined cDNA sequence for JP8E10

SEQ ID NO: 301 is the determined cDNA sequence for JP8F9

SEQ ID NO: 302 is the determined cDNA sequence for JP8H9

SEQ ID NO: 303 is the determined cDNA sequence for JP8C12

SEQ ID NO: 304 is the determined cDNA sequence for JP8E11

SEQ ID NO: 305 is the determined cDNA sequence for JP8E12

SEQ ID NO: 306 is the amino acid sequence for the peptide PS2#12

SEQ ID NO: 307 is the determined cDNA sequence for P711P

SEQ ID NO: 308 is the determined cDNA sequence for P712P

SEQ ID NO: 309 is the determined cDNA sequence for CLONE23

SEQ ID NO: 310 is the determined cDNA sequence for P774P

SEQ ID NO: 311 is the determined cDNA sequence for P775P

SEQ ID NO: 312 is the determined cDNA sequence for P715P

SEQ ID NO: 313 is the determined cDNA sequence for P710P

SEQ ID NO: 314 is the determined cDNA sequence for P767P

SEQ ID NO: 315 is the determined cDNA sequence for P768P

SEQ ID NO: 316-325 are the determined cDNA sequences of previouslyisolated genes

SEQ ID NO: 326 is the determined cDNA sequence for P703PDE5

SEQ ID NO: 327 is the predicted amino acid sequence for P703PDE5

SEQ ID NO: 328 is the determined cDNA sequence for P703P6.26

SEQ ID NO: 329 is the predicted amino acid sequence for P703P6.26

SEQ ID NO: 330 is the determined cDNA sequence for P703PX-23

SEQ ID NO: 331 is the predicted amino acid sequence for P703PX-23

SEQ ID NO: 332 is the determined full length cDNA sequence for P509S

SEQ ID NO: 333 is the determine d extended cDNA sequence for P707P (alsoreferred to as 11-C9)

SEQ ID NO: 334 is the determined cDNA sequence for P714P

SEQ ID NO: 335 is the determined cDNA sequence for P705 P (also referredto as 9-F3)

SEQ ID NO: 336 is the predicted amino acid sequence for P705P

SEQ ID NO: 337 is the amino acid sequence of the peptide P1S#10

SEQ ID NO: 338 is the amino acid sequence of the peptide p5

SEQ ID NO: 339 is the predicted amino acid sequence of P509S

SEQ ID NO: 340 is the determined cDNA sequence for P778P

SEQ ID NO: 341 is the determined cDNA sequence for P786P

SEQ ID NO: 342 is the determined cDNA sequence for P789P

SEQ ID NO: 343 is the determined cDNA sequence for a clone showinghomology to Homo sapiens MM46 mRNA

SEQ ID NO: 344 is the determined cDNA sequence for a clone showinghomology to Homo sapiens TNF-alpha stimulated ABC protein (ABC50) mRNA

SEQ ID NO: 345 is the determined cDNA sequence for a clone showinghomology to Homo sapiens mRNA for E-cadherin

SEQ ID NO: 346 is the determined cDNA sequence for a clone showinghomology to Human nuclear-encoded mitochondrial serinehydroxymethyltransferase (SHMT)

SEQ ID NO: 347 is the determined cDNA sequence for a clone showinghomology to Homo sapiens natural resistance-associated macrophageprotein2 (NRAMP2)

SEQ ID NO: 348 is the determined cDNA sequence for a clone showinghomology to Homo sapiens phosphoglucomutase-related protein (PGMRP)

SEQ ID NO: 349 is the determined cDNA sequence for a clone showinghomology to Human mRNA for proteosome subunit p40

SEQ ID NO: 350 is the determined cDNA sequence for P777P

SEQ ID NO: 351 is the determined cDNA sequence for P779P

SEQ ID NO: 352 is the determined cDNA sequence for P790P

SEQ ID NO: 353 is the determined cDNA sequence for P784P

SEQ ID NO: 354 is the determined cDNA sequence for P776P

SEQ ID NO: 355 is the determined cDNA sequence for P780P

SEQ ID NO: 356 is the determined cDNA sequence for P544S

SEQ ID NO: 357 is the determined cDNA sequence for P745S

SEQ ID NO: 358 is the determined cDNA sequence for P782P

SEQ ID NO: 359 is the determined cDNA sequence for P783P

SEQ ID NO: 360 is the determined cDNA sequence for unknown 17984

SEQ ID NO: 361 is the determined cDNA sequence for P787P

SEQ ID NO: 362 is the determined cDNA sequence for P788P

SEQ ID NO: 363 is the determined cDNA sequence for unknown 17994

SEQ ID NO: 364 is the determined cDNA sequence for P781P

SEQ ID NO: 365 is the determined cDNA sequence for P785P

SEQ ID NO: 366-375 are the determined cDNA sequences for splice variantsof B305D.

SEQ ID NO: 376 is the predicted amino acid sequence encoded by thesequence of SEQ ID NO: 366.

SEQ ID NO: 377 is the predicted amino acid sequence encoded by thesequence of SEQ ID NO: 372.

SEQ ID NO: 378 is the predicted amino acid sequence encoded by thesequence of SEQ ID NO: 373.

SEQ ID NO: 379 is the predicted amino acid sequence encoded by thesequence of SEQ ID NO: 374.

SEQ ID NO: 380 is the predicted amino acid sequence encoded by thesequence of SEQ ID NO: 375.

SEQ ID NO: 381 is the determined cDNA sequence for B716P.

SEQ ID NO: 382 is the determined full-length cDNA sequence for P711P.

SEQ ID NO: 383 is the predicted amino acid sequence for P711P.

SEQ ID NO: 384 is the cDNA sequence for P1000C.

SEQ ID NO: 385 is the cDNA sequence for CGI-82.

SEQ ID NO:386 is the cDNA sequence for 23320.

SEQ ID NO:387 is the cDNA sequence for CGI-69.

SEQ ID NO:388 is the cDNA sequence for L-iditol-2-dehydrogenase.

SEQ ID NO:389 is the cDNA sequence for 23379.

SEQ ID NO:390 is the cDNA sequence for 23381.

SEQ ID NO:391 is the cDNA sequence for KIAA0122.

SEQ ID NO:392 is the cDNA sequence for 23399.

SEQ ID NO:393 is the cDNA sequence for a previously identified gene.

SEQ ID NO:394 is the cDNA sequence for HCLBP.

SEQ ID NO:395 is the cDNA sequence for transglutaminase.

SEQ ID NO:396 is the cDNA sequence for a previously identified gene.

SEQ ID NO:397 is the cDNA sequence for PAP.

SEQ ID NO:398 is the cDNA sequence for Ets transcription factor PDEF.

SEQ ID NO:399 is the cDNA sequence for hTGR.

SEQ ID NO:400 is the cDNA sequence for KIAA0295.

SEQ ID NO:401 is the cDNA sequence for 22545.

SEQ ID NO:402 is the cDNA sequence for 22547.

SEQ ID NO:403 is the cDNA sequence for 22548.

SEQ ID NO:404 is the cDNA sequence for 22550.

SEQ ID NO:405 is the cDNA sequence for 22551.

SEQ ID NO:406 is the cDNA sequence for 22552.

SEQ ID NO:407 is the cDNA sequence for 22553 (also known as P1020C).

SEQ ID NO:408 is the cDNA sequence for 22558.

SEQ ID NO:409 is the cDNA sequence for 22562.

SEQ ID NO:410 is the cDNA sequence for 22565.

SEQ ID NO:411 is the cDNA sequence for 22567.

SEQ ID NO:412 is the cDNA sequence for 22568.

SEQ ID NO:413 is the cDNA sequence for 22570.

SEQ ID NO:414 is the cDNA sequence for 22571.

SEQ ID NO:415 is the cDNA sequence for 22572.

SEQ ID NO:416 is the cDNA sequence for 22573.

SEQ ID NO:417 is the cDNA sequence for 22573.

SEQ ID NO:418 is the cDNA sequence for 22575.

SEQ ID NO:419 is the cDNA sequence for 22580.

SEQ ID NO:420 is the cDNA sequence for 22581.

SEQ ID NO:421 is the cDNA sequence for 22582.

SEQ ID NO:422 is the cDNA sequence for 22583.

SEQ ID NO:423 is the cDNA sequence for 22584.

SEQ ID NO:424 is the cDNA sequence for 22585.

SEQ ID NO:425 is the cDNA sequence for 22586.

SEQ ID NO:426 is the cDNA sequence for 22587.

SEQ ID NO:427 is the cDNA sequence for 22588.

SEQ ID NO:428 is the cDNA sequence for 22589.

SEQ ID NO:429 is the cDNA sequence for 22590.

SEQ ID NO:430 is the cDNA sequence for 22591.

SEQ ID NO:431 is the cDNA sequence for 22592.

SEQ ID NO:432 is the cDNA sequence for 22593.

SEQ ID NO:433 is the cDNA sequence for 22594.

SEQ ID NO:434 is the cDNA sequence for 22595.

SEQ ID NO:435 is the cDNA sequence for 22596.

SEQ ID NO:436 is the cDNA sequence for 22847.

SEQ ID NO:437 is the cDNA sequence for 22848.

SEQ ID NO:438 is the cDNA sequence for 22849.

SEQ ID NO:439 is the cDNA sequence for 22851.

SEQ ID NO:440 is the cDNA sequence for 22852.

SEQ ID NO:441 is the cDNA sequence for 22853.

SEQ ID NO:442 is the cDNA sequence for 22854.

SEQ ID NO:443 is the cDNA sequence for 22855.

SEQ ID NO:444 is the cDNA sequence for 22856.

SEQ ID NO:445 is the cDNA sequence for 22857.

SEQ ID NO:446 is the cDNA sequence for 23601.

SEQ ID NO:447 is the cDNA sequence for 23602.

SEQ ID NO:448 is the cDNA sequence for 23605.

SEQ ID NO:449 is the cDNA sequence for 23606.

SEQ ID NO:450 is the cDNA sequence for 23612.

SEQ ID NO:451 is the cDNA sequence for 23614.

SEQ ID NO:452 is the cDNA sequence for 23618.

SEQ ID NO:453 is the cDNA sequence for 23622.

SEQ ID NO:454 is the cDNA sequence for folate hydrolase.

SEQ ID NO:455 is the cDNA sequence for LIM protein.

SEQ ID NO:456 is the cDNA sequence for a known gene.

SEQ ID NO:457 is the cDNA sequence for a known gene.

SEQ ID NO:458 is the cDNA sequence for a previously identified gene.

SEQ ID NO:459 is the cDNA sequence for 23045.

SEQ ID NO:460 is the cDNA sequence for 23032.

SEQ ID NO:461 is the cDNA sequence for 23054.

SEQ ID NO:462-467 are cDNA sequences for known genes.

SEQ ID NO:468-471 are cDNA sequences for P710P.

SEQ ID NO:472 is a cDNA sequence for P1001C.

SEQ ID NO: 473 is the determined cDNA sequence for a first splicevariant of P775P (referred to as 27505).

SEQ ID NO: 474 is the determined cDNA sequence for a second splicevariant of P775P (referred to as 19947).

SEQ ID NO: 475 is the determined cDNA sequence for a third splicevariant of P775P (referred to as 19941).

SEQ ID NO: 476 is the determined cDNA sequence for a fourth splicevariant of P775P (referred to as 19937).

SEQ ID NO: 477 is a first predicted amino acid sequence encoded by thesequence of SEQ ID NO: 474.

SEQ ID NO: 478 is a second predicted amino acid sequence encoded by thesequence of SEQ ID NO: 474.

SEQ ID NO: 479 is the predicted amino acid sequence encoded by thesequence of SEQ ID NO: 475.

SEQ ID NO: 480 is a first predicted amino acid sequence encoded by thesequence of SEQ ID NO: 473.

SEQ ID NO: 481 is a second predicted amino acid sequence encoded by thesequence of SEQ ID NO: 473.

SEQ ID NO: 482 is a third predicted amino acid sequence encoded by thesequence of SEQ ID NO: 473.

SEQ ID NO: 483 is a fourth predicted amino acid sequence encoded by thesequence of SEQ ID NO: 473.

SEQ ID NO: 484 is the first 30 amino acids of the M. tuberculosisantigen Ra12.

SEQ ID NO: 485 is the PCR primer AW025.

SEQ ID NO: 486 is the PCR primer AW003.

SEQ ID NO: 487 is the PCR primer AW027.

SEQ ID NO: 488 is the PCR primer AW026.

SEQ ID NO: 489-501 are peptides employed in epitope mapping studies.

SEQ ID NO: 502 is the determined cDNA sequence of the complementaritydetermining region for the anti-P503S monoclonal antibody 20D4.

SEQ ID NO: 503 is the determined cDNA sequence of the complementaritydetermining region for the anti-P503S monoclonal antibody JA1.

SEQ ID NO: 504 & 505 are peptides employed in epitope mapping studies.

SEQ ID NO: 506 is the determined cDNA sequence of the complementaritydetermining region for the anti-P703P monoclonal antibody 8H2.

SEQ ID NO: 507 is the determined cDNA sequence of the complementaritydetermining region for the anti-P703P monoclonal antibody 7H8.

SEQ ID NO: 508 is the determined cDNA sequence of the complementaritydetermining region for the anti-P703P monoclonal antibody 2D4.

SEQ ID NO: 509-522 are peptides employed in epitope mapping studies.

SEQ ID NO: 523 is a mature form of P703P used to raise antibodiesagainst P703P.

SEQ ID NO: 524 is the putative full-length cDNA sequence of P703P.

SEQ ID NO: 525 is the predicted amino acid sequence encoded by SEQ IDNO: 524.

SEQ ID NO: 526 is the full-length cDNA sequence for P790P.

SEQ ID NO: 527 is the predicted amino acid sequence for P790P.

SEQ ID NO: 528 & 529 are PCR primers.

SEQ ID NO: 530 is the cDNA sequence of a splice variant of SEQ ID NO:366.

SEQ ID NO: 531 is the cDNA sequence of the open reading frame of SEQ DNO: 530.

SEQ ID NO: 532 is the predicted amino acid encoded by the sequence ofSEQ ID NO: 531.

SEQ ID NO: 533 is the DNA sequence of a putative ORF of P775P.

SEQ ID NO: 534 is the predicted amino acid sequence encoded by SEQ IDNO: 533.

SEQ ID NO: 535 is a first full-length cDNA sequence for P510S.

SEQ ID NO: 536 is a second full-length cDNA sequence for P510S.

SEQ ID NO: 537 is the predicted amino acid sequence encoded by SEQ IDNO: 535.

SEQ ID NO: 538 is the predicted amino acid sequence encoded by SEQ IDNO: 536.

SEQ ID NO: 539 is the peptide P501S-370.

SEQ ID NO: 540 is the peptide P501S-376.

SEQ ID NO: 541-551 are epitopes of P501S.

SEQ ID NO: 552 is an extended cDNA sequence for P712P.

SEQ ID NO: 553-568 are the amino acid sequences encoded by predictedopen reading frames within SEQ ID NO: 552.

SEQ ID NO: 569 is an extended cDNA sequence for P776P.

SEQ ID NO: 570 is the determined cDNA sequence for a splice variant ofP776P referred to as contig 6.

SEQ ID NO: 571 is the determined cDNA sequence for a splice variant ofP776P referred to as contig 7.

SEQ ID NO: 572 is the determined cDNA sequence for a splice variant ofP776P referred to as contig 14.

SEQ ID NO: 573 is the amino acid sequence encoded by a first predictedORF of SEQ ID NO: 570.

SEQ ID NO: 574 is the amino acid sequence encoded by a second predictedORF of SEQ ID NO: 570.

SEQ ID NO: 575 is the amino acid sequence encoded by a predicted ORF ofSEQ ID NO: 571.

SEQ ID NO: 576-586 are amino acid sequences encoded by predicted ORFs ofSEQ ID NO: 569.

SEQ ID NO: 587 is a DNA consensus sequence of the sequences of P767P andP777P.

SEQ ID NO: 588-590 are amino acid sequences encoded by predicted ORFs ofSEQ ID NO: 587.

SEQ ID NO: 591 is an extended cDNA sequence for P1020C.

SEQ ID NO: 592 is the predicted amino acid sequence encoded by thesequence of SEQ ID NO: P1020C.

SEQ ID NO: 593 is a splice variant of P775P referred to as 50748.

SEQ ID NO: 594 is a splice variant of P775P referred to as 50717.SEQ IDNO: 595 is a splice variant of P775P referred to as 45985.

SEQ ID NO: 596 is a splice variant of P775P referred to as 38769.

SEQ ID NO: 597 is a splice variant of P775P referred to as 37922.

SEQ ID NO: 598 is a splice variant of P510S referred to as 49274.

SEQ ID NO: 599 is a splice variant of P510S referred to as 39487.

SEQ ID NO: 600 is a splice variant of P504S referred to as 5167.16.

SEQ ID NO: 601 is a splice variant of P504S referred to as 5167.1.

SEQ ID NO: 602 is a splice variant of P504S referred to as 5163.46.

SEQ ID NO: 603 is a splice variant of P504S referred to as 5163.42.

SEQ ID NO: 604 is a splice variant of P504S referred to as 5163.34.

SEQ ID NO: 605 is a splice variant of P504S referred to as 5163.17.

SEQ ID NO: 606 is a splice variant of P501S referred to as 10640.

SEQ ID NO: 607-615 are the sequences of PCR primers.

SEQ ID NO: 616 is the determined cDNA sequence of a fusion of P703P andPSA.

SEQ ID NO: 617 is the amino acid sequence of the fusion of P703P andPSA.

SEQ ID NO: 618-689 are determined cDNA sequences of prostate-specificclones.

SEQ ID NO: 690 is the cDNA sequence of the gene DD3.

SEQ ID NO: 691-697 are determined cDNA sequences of prostate-specificclones.

SEQ ID NO: 698 is an extended cDNA sequence for P714P.

SEQ ID NO: 699-701 are the cDNA sequences for splice variants of P704P.

SEQ ID NO: 702 is the cDNA sequence of a spliced variant of P553Sreferred to as P553S-14.

SEQ ID NO: 703 is the cDNA sequence of a spliced variant of P553Sreferred to as P553S-12.

SEQ ID NO: 704 is the cDNA sequence of a spliced variant of P553Sreferred to as P553S-10.

SEQ ID NO: 705 is the cDNA sequence of a spliced variant of P553Sreferred to as P553S-6.

SEQ ID NO: 706 is the amino acid sequence encoded by SEQ ID NO: 705.

SEQ ID NO: 707 is the amino acid sequence encoded by SEQ ID NO: 702 SEQID NO: 708 is a second amino acid sequence encoded by SEQ ID NO: 702.

SEQ ID NO: 709-772 are determined cDNA sequences of prostate-specificclones.

SEQ ID NO: 773 is a first full-length cDNA sequence forprostate-specific transglutaminase gene (also referred to herein asP558S).

SEQ ID NO: 774 is a second full-length cDNA sequence forprostate-specific transglutaminase gene.

SEQ ID NO: 775 is the amino acid sequence encoded by the sequence of SEQID NO: 773.

SEQ ID NO: 776 is the amino acid sequence encoded by the sequence of SEQID NO: 774.

SEQ ID NO: 777 is the full-length cDNA sequence for P788P.

SEQ ID NO: 778 is the amino acid sequence encoded by SEQ ID NO: 777.

SEQ ID NO: 779 is the determined cDNA sequence for a polymorphic variantof P788P.

SEQ ID NO: 780 is the amino acid sequence encoded by SEQ ID NO: 779.

SEQ ID NO: 781 is the amino acid sequence of peptide 4 from P703P.

SEQ ID NO: 782 is the cDNA sequence that encodes peptide 4 from P703P.

SEQ ID NO: 783-798 are the cDNA sequence encoding epitopes of P703P.

SEQ ID NO: 799-814 are the amino acid sequences of epitopes of P703P.

SEQ ID NO: 815 and 816 are PCR primers.

SEQ ID NO: 817 is the cDNA sequence encoding an N-terminal portion ofP788P expressed in E. coli.

SEQ ID NO: 818 is the amino acid sequence of the N-terminal portion ofP788P expressed in E. coli.

SEQ ID NO: 819 is the amino acid sequence of the M. tuberculosis antigenRa12.

SEQ ID NO: 820 and 821 are PCR primers.

SEQ ID NO: 822 is the cDNA sequence for the Ra12-P510S-C construct.

SEQ ID NO: 823 is the cDNA sequence for the P510S-C construct.

SEQ ID NO: 824 is the cDNA sequence for the P510S-E3 construct.

SEQ ID NO: 825 is the amino acid sequence for the Ra12-P510S-Cconstruct.

SEQ ID NO: 826 is the amino acid sequence for the P510S-C construct.

SEQ ID NO: 827 is the amino acid sequence for the P510S-E3 construct.

SEQ ID NO: 828-833 are PCR primers. SEQ ID NO: 834 is the cDNA sequenceof the construct Ra12-P775P-ORF3.

SEQ ID NO: 835 is the amino acid sequence of the constructRa12-P775P-ORF3.

SEQ ID NO: 836 and 837 are PCR primers.

SEQ ID NO: 838 is the determined amino acid sequence for a P703P His tagfusion protein.

SEQ ID NO: 839 is the determined cDNA sequence for a P703P His tagfusion protein.

SEQ ID NO: 840 and 841 are PCR primers.

SEQ ID NO: 842 is the determined amino acid sequence for a P705P His tagfusion protein.

SEQ ID NO: 843 is the determined cDNA sequence for a P705P His tagfusion protein.

SEQ ID NO: 844 and 845 are PCR primers.

SEQ ID NO: 846 is the determined amino acid sequence for a P711P His tagfusion protein.

SEQ ID NO: 847 is the determined cDNA sequence for a P711P His tagfusion protein.

SEQ ID NO: 848 is the amino acid sequence of the M. tuberculosis antigenRa12.

SEQ ID NO: 849 and 850 are PCR primers.

SEQ ID NO: 851 is the determined cDNA sequence for the constructRa12-P501S-E2.

SEQ ID NO: 852 is the determined amino acid sequence for the constructRa12-P501S-E2.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed tocompositions and methods for using the compositions, for example in thetherapy and diagnosis of cancer, such as prostate cancer. Certainillustrative compositions described herein include prostate-specificpolypeptides, polynucleotides encoding such polypeptides, binding agentssuch as antibodies, antigen presenting cells (APCs) and/or immune systemcells (e.g., T cells). A “prostate-specific protein,” as the term isused herein, refers generally to a protein that is expressed in prostatecells at a level that is at least two fold, and preferably at least fivefold, greater than the level of expression in other normal tissues, asdetermined using a representative assay provided herein. Certainprostate-specific proteins are tumor proteins that react detectably(within an immunoassay, such as an ELISA or Western blot) with antiseraof a patient afflicted with prostate cancer.

Therefore, in accordance with the above, and as described further below,the present invention provides illustrative polynucleotide compositionshaving sequences set forth in SEQ ID NO: 1-111, 115-171, 173-175, 177,179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476,524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606,618-705, 709-774, 777, 789, 817, 823 and 824, illustrative polypeptidecompositions having amino acid sequences set forth in SEQ ID NO:112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383, 477-483,496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551, 553-568,573-586, 588-590, 592, 706-708, 775, 776, 778, 780, 781, 811, 814, 818,826 and 827, antibody compositions capable of binding such polypeptides,and numerous additional embodiments employing such compositions, forexample in the detection, diagnosis and/or therapy of human prostatecancer.

Polynucleotide Compositions

As used herein, the terms “DNA segment” and “polynucleotide” refer to aDNA molecule that has been isolated free of total genomic DNA of aparticular species. Therefore, a DNA segment encoding a polypeptiderefers to a DNA segment that contains one or more coding sequences yetis substantially isolated away from, or purified free from, totalgenomic DNA of the species from which the DNA segment is obtained.Included within the terms “DNA segment” and “polynucleotide” are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phagemids, phage,viruses, and the like.

As will be understood by those skilled in the art, the DNA segments ofthis invention can include genomic sequences, extra-genomic andplasmid-encoded sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides, peptidesand the like. Such segments may be naturally isolated, or modifiedsynthetically by the hand of man.

“Isolated,” as used herein, means that a polynucleotide is substantiallyaway from other coding sequences, and that the DNA segment does notcontain large portions of unrelated coding DNA, such as largechromosomal fragments or other functional genes or polypeptide codingregions. Of course, this refers to the DNA segment as originallyisolated, and does not exclude genes or coding regions later added tothe segment by the hand of man.

As will be recognized by the skilled artisan, polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. RNA molecules includeHNRNA molecules, which contain introns and correspond to a DNA moleculein a one-to-one manner, and mRNA molecules, which do not containintrons. Additional coding or non-coding sequences may, but need not, bepresent within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a prostate-specific protein or a portion thereof)or may comprise a variant, or a biological or antigenic functionalequivalent of such a sequence. Polynucleotide variants may contain oneor more substitutions, additions, deletions and/or insertions, asfurther described below, preferably such that the immunogenicity of theencoded polypeptide is not diminished, relative to a native tumorprotein. The effect on the immunogenicity of the encoded polypeptide maygenerally be assessed as described herein. The term “variants” alsoencompasses homologous genes of xenogenic origin.

When comparing polynucleotide or polypeptide sequences, two sequencesare said to be “identical” if the sequence of nucleotides or amino acidsin the two sequences is the same when aligned for maximumcorrespondence, as described below. Comparisons between two sequencesare typically performed by comparing the sequences over a comparisonwindow to identify and compare local regions of sequence similarity. A“comparison window” as used herein, refers to a segment of at leastabout 20 contiguous positions, usually 30 to about 75, 40 to about 50,in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides and polypeptides of the invention.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. In one illustrativeexample, cumulative scores can be calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix can be used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, andexpectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff andHenikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of50, expectation (E) of 10, M=5, N=4 and a comparison of both strands.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e., the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Therefore, the present invention encompasses polynucleotide andpolypeptide sequences having substantial identity to the sequencesdisclosed herein, for example those comprising at least 50% sequenceidentity, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to apolynucleotide or polypeptide sequence of this invention using themethods described herein, (e.g., BLAST analysis using standardparameters, as described below). One skilled in this art will recognizethat these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like.

In additional embodiments, the present invention provides isolatedpolynucleotides and polypeptides comprising various lengths ofcontiguous stretches of sequence identical to or complementary to one ormore of the sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise at least about 15, 20, 30, 40,50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguousnucleotides of one or more of the sequences disclosed herein as well asall intermediate lengths there between. It will be readily understoodthat “intermediate lengths”, in this context, means any length betweenthe quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30,31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151,152, 153, etc.; including all integers through 200-500; 500-1,000, andthe like.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, illustrative DNA segments withtotal lengths of about 10,000, about 5000, about 3000, about 2,000,about 1,000, about 500, about 200, about 100, about 50 base pairs inlength, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

In other embodiments, the present invention is directed topolynucleotides that are capable of hybridizing under moderatelystringent conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridizing at 50° C.-65° C., 5×SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2×,0.5×and 0.2×SSC containing 0.1% SDS.

Moreover, it will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention. Further, alleles of the genes comprising thepolynucleotide sequences provided herein are within the scope of thepresent invention. Alleles are endogenous genes that are altered as aresult of one or more mutations, such as deletions, additions and/orsubstitutions of nucleotides. The resulting mRNA and protein may, butneed not, have an altered structure or function. Alleles may beidentified using standard techniques (such as hybridization,amplification and/or database sequence comparison).

Probes and Primers

In other embodiments of the present invention, the polynucleotidesequences provided herein can be advantageously used as probes orprimers for nucleic acid hybridization. As such, it is contemplated thatnucleic acid segments that comprise a sequence region of at least about15 nucleotide long contiguous sequence that has the same sequence as, oris complementary to, a 15 nucleotide long contiguous sequence disclosedherein will find particular utility. Longer contiguous identical orcomplementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200,500, 1000 (including all intermediate lengths) and even up to fulllength sequences will also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize to asequence of interest will enable them to be of use in detecting thepresence of complementary sequences in a given sample. However, otheruses are also envisioned, such as the use of the sequence informationfor the preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

Polynucleotide molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so (including intermediate lengths as well),identical or complementary to a polynucleotide sequence disclosedherein, are particularly contemplated as hybridization probes for usein, e.g., Southern and Northern blotting. This would allow a geneproduct, or fragment thereof, to be analyzed, both in diverse cell typesand also in various bacterial cells. The total size of fragment, as wellas the size of the complementary stretch(es), will ultimately depend onthe intended use or application of the particular nucleic acid segment.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 15 and about 100 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

The use of a hybridization probe of about 15-25 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 15 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 25 contiguous nucleotides,or even longer where desired.

Hybridization probes may be selected from any portion of any of thesequences disclosed herein. All that is required is to review thesequence set forth in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305,307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524,526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705,709-774, 777, 789, 817, 823 and 824, or to any continuous portion of thesequence, from about 15-25 nucleotides in length up to and including thefull length sequence, that one wishes to utilize as a probe or primer.The choice of probe and primer sequences may be governed by variousfactors. For example, one may wish to employ primers from towards thetermini of the total sequence.

Small polynucleotide segments or fragments may be readily prepared by,for example, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. No. 4,683,202(incorporated herein by reference), by introducing selected sequencesinto recombinant vectors for recombinant production, and by otherrecombinant DNA techniques generally known to those of skill in the artof molecular biology.

The nucleotide sequences of the invention may be used for their abilityto selectively form duplex molecules with complementary stretches of theentire gene or gene fragments of interest. Depending on the applicationenvisioned, one will typically desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by a salt concentration of fromabout 0.02 M to about 0.15 M salt at temperatures of from about 50° C.to about 70° C. Such selective conditions tolerate little, if any,mismatch between the probe and the template or target strand, and wouldbe particularly suitable for isolating related sequences.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template, less stringent (reduced stringency) hybridizationconditions will typically be needed in order to allow formation of theheteroduplex. In these circumstances, one may desire to employ saltconditions such as those of from about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Cross-hybridizingspecies can thereby be readily identified as positively hybridizingsignals with respect to control hybridizations. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

Polynucleotide Identification and Characterization

Polynucleotides may be identified, prepared and/or manipulated using anyof a variety of well established techniques. For example, apolynucleotide may be identified, as described in more detail below, byscreening a microarray of cDNAs for tumor-associated expression (i.e.,expression that is at least two fold greater in a tumor than in normaltissue, as determined using a representative assay provided herein).Such screens may be performed, for example, using a Synteni microarray(Palo Alto, Calif.) according to the manufacturer's instructions (andessentially as described by Schena et al., Proc. Natl. Acad. Sci. USA93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA94:2150-2155, 1997). Alternatively, polynucleotides may be amplifiedfrom cDNA prepared from cells expressing the proteins described herein,such as prostate-specific cells. Such polynucleotides may be amplifiedvia polymerase chain reaction (PCR). For this approach,sequence-specific primers may be designed based on the sequencesprovided herein, and may be purchased or synthesized.

An amplified portion of a polynucleotide of the present invention may beused to isolate a full length gene from a suitable library (e.g., aprostate tumor cDNA library) using well known techniques. Within suchtechniques, a library (cDNA or genomic) is screened using one or morepolynucleotide probes or primers suitable for amplification. Preferably,a library is size-selected to include larger molecules. Random primedlibraries may also be preferred for identifying 5′ and upstream regionsof genes. Genomic libraries are preferred for obtaining introns andextending 5′ sequences.

For hybridization techniques, a partial sequence may be labeled (e.g.,by nick-translation or end-labeling with ³²p) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. cDNA clones may be analyzed to determine the amount ofadditional sequence by, for example, PCR using a primer from the partialsequence and a primer from the vector. Restriction maps and partialsequences may be generated to identify one or more overlapping clones.The complete sequence may then be determined using standard techniques,which may involve generating a series of deletion clones. The resultingoverlapping sequences can then assembled into a single contiguoussequence. A full length cDNA molecule can be generated by ligatingsuitable fragments, using well known techniques.

Alternatively, there are numerous amplification techniques for obtaininga full length coding sequence from a partial cDNA sequence. Within suchtechniques, amplification is generally performed via PCR. Any of avariety of commercially available kits may be used to perform theamplification step. Primers may be designed using, for example, softwarewell known in the art. Primers are preferably 22-30 nucleotides inlength, have a GC content of at least 50% and anneal to the targetsequence at temperatures of about 68° C. to 72° C. The amplified regionmay be sequenced as described above, and overlapping sequences assembledinto a contiguous sequence.

One such amplification technique is inverse PCR (see Triglia et al.,Nucl. Acids Res. 16:8186, 1988), which uses restriction enzymes togenerate a fragment in the known region of the gene. The fragment isthen circularized by intramolecular ligation and used as a template forPCR with divergent primers derived from the known region. Within analternative approach, sequences adjacent to a partial sequence may beretrieved by amplification with a primer to a linker sequence and aprimer specific to a known region. The amplified sequences are typicallysubjected to a second round of amplification with the same linker primerand a second primer specific to the known region. A variation on thisprocedure, which employs two primers that initiate extension in oppositedirections from the known sequence, is described in WO 96/38591. Anothersuch technique is known as “rapid amplification of cDNA ends” or RACE.This technique involves the use of an internal primer and an externalprimer, which hybridizes to a polyA region or vector sequence, toidentify sequences that are 5′ and 3′ of a known sequence. Additionaltechniques include capture PCR (Lagerstrom et al., PCR Methods Applic.1:111-19, 1991) and walking PCR (Parker et al., Nucl. Acids. Res.19:3055-60, 1991). Other methods employing amplification may also beemployed to obtain a full length cDNA sequence.

In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

Polynucleotide Expression in Host Cells

In other embodiments of the invention, polynucleotide sequences orfragments thereof which encode polypeptides of the invention, or fusionproteins or functional equivalents thereof, may be used in recombinantDNA molecules to direct expression of a polypeptide in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other DNAsequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and these sequences maybe used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. For example, DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

Sequences encoding a desired polypeptide may be synthesized, in whole orin part, using chemical methods well known in the art (see Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al.(1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the proteinitself may be produced using chemical methods to synthesize the aminoacid sequence of a polypeptide, or a portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(Roberge, J. Y. et al. (1995) Science 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431 A PeptideSynthesizer (Perkin Elmer, Palo Alto, Calif.).

A newly synthesized peptide may be substantially purified by preparativehigh performance liquid chromatography (e.g., Creighton, T. (1983)Proteins, Structures and Molecular Principles, WH Freeman and Co., NewYork, N.Y.) or other comparable techniques available in the art. Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure).Additionally, the amino acid sequence of a polypeptide, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

In order to express a desired polypeptide, the nucleotide sequencesencoding the polypeptide, or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook, J. et al.(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York. N.Y.

A variety of expression vector/host systems may be utilized to containand express polynucleotide sequences. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are needed, for example for the inductionof antibodies, vectors which direct high level expression of fusionproteins that are readily purified may be used. Such vectors include,but are not limited to, the multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of .beta.-galactosidase so that a hybrid proteinis produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega,Madison, Wis.) may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhard,E. K. et al. (1994) Proc. Natl. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc.Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylationglycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) geneswhich can be employed in tk.sup.- or aprt.sup.- cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, beta-glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include membrane, solution, or chipbased technologies for the detection and/or quantification of nucleicacid or protein.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on a given polypeptide may be preferred forsome applications, but a competitive binding assay may also be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul. Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3:263-281) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.12:441-453).

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield J. (1963) J. Am.Chem. Soc. 85:2149-2154). Protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431 A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

Site-Specific Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent polypeptides,through specific mutagenesis of the underlying polynucleotides thatencode them. The technique, well-known to those of skill in the art,further provides a ready ability to prepare and test sequence variants,for example, incorporating one or more of the foregoing considerations,by introducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments of the present invention, the inventorscontemplate the mutagenesis of the disclosed polynucleotide sequences toalter one or more properties of the encoded polypeptide, such as theantigenicity of a polypeptide vaccine. The techniques of site-specificmutagenesis are well-known in the art, and are widely used to createvariants of both polypeptides and polynucleotides. For example,site-specific mutagenesis is often used to alter a specific portion of aDNA molecule. In such embodiments, a primer comprising typically about14 to about 25 nucleotides or so in length is employed, with about 5 toabout 10 residues on both sides of the junction of the sequence beingaltered.

As will be appreciated by those of skill in the art, site-specificmutagenesis techniques have often employed a phage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage are readily commercially-available and their use isgenerally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982, each incorporated herein byreference, for that purpose.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

Polynucleotide Amplification Techniques

A number of template dependent processes are available to amplify thetarget sequences of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR™) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, each of which is incorporated herein by reference in itsentirety. Briefly, in PCR™, two primer sequences are prepared which arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase (e.g., Taq polymerase). Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe target sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction product and the process isrepeated. Preferably reverse transcription and PCR™ amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

Another method for amplification is the ligase chain reaction (referredto as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308 (specificallyincorporated herein by reference in its entirety). In LCR, twocomplementary probe pairs are prepared, and in the presence of thetarget sequence, each pair will bind to opposite complementary strandsof the target such that they abut. In the presence of a ligase, the twoprobe pairs will link to form a single unit. By temperature cycling, asin PCR™, bound ligated units dissociate from the target and then serveas “target sequences” for ligation of excess probe pairs. U.S. Pat. No.4,883,750, incorporated herein by reference in its entirety, describesan alternative method of amplification similar to LCR for binding probepairs to a target sequence.

Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No.PCT/US87/00880, incorporated herein by reference in its entirety, mayalso be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA that has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[α-thio]triphosphates in one strand of arestriction site (Walker et al., 1992, incorporated herein by referencein its entirety), may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e. nick translation. Asimilar method, called Repair Chain Reaction (RCR) is another method ofamplification which may be usefuil in the present invention and isinvolves annealing several probes throughout a region targeted foramplification, followed by a repair reaction in which only two of thefour bases are present. The other two bases can be added as biotinylatedderivatives for easy detection. A similar approach is used in SDA.

Sequences can also be detected using a cyclic probe reaction (CPR). InCPR, a probe having a 3′ and 5′ sequences of non-target DNA and aninternal or “middle” sequence of the target protein specific RNA ishybridized to DNA which is present in a sample. Upon hybridization, thereaction is treated with RNaseH, and the products of the probe areidentified as distinctive products by generating a signal that isreleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated. Thus, CPR involvesamplifying a signal generated by hybridization of a probe to a targetgene specific expressed nucleic acid.

Still other amplification methods described in Great Britain Pat. Appl.No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No. PCT/US89/01025,each of which is incorporated herein by reference in its entirety, maybe used in accordance with the present invention. In the formerapplication, “modified” primers are used in a PCR-like, template andenzyme dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes is added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS) (Kwoh et al., 1989; PCT Intl. Pat. Appl.Publ. No. WO 88/10315, incorporated herein by reference in itsentirety), including nucleic acid sequence based amplification (NASBA)and 3SR. In NASBA, the nucleic acids can be prepared for amplificationby standard phenol/chloroforrn extraction, heat denaturation of asample, treatment with lysis buffer and minispin columns for isolationof DNA and RNA or guanidinium chloride extraction of RNA. Theseamplification techniques involve annealing a primer that has sequencesspecific to the target sequence. Following polymerization, DNA/RNAhybrids are digested with RNase H while double stranded DNA moleculesare heat-denatured again. In either case the single stranded DNA is madefully double stranded by addition of second target-specific primer,followed by polymerization. The double stranded DNA molecules are thenmultiply transcribed by a polymerase such as T7 or SP6. In an isothermalcyclic reaction, the RNAs are reverse transcribed into DNA, andtranscribed once again with a polymerase such as T7 or SP6. Theresulting products, whether truncated or complete, indicatetarget-specific sequences.

Eur. Pat. Appl. Publ. No. 329,822, incorporated herein by reference inits entirety, disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from resultingDNA:RNA duplex by the action of ribonuclease H (RNase H, an RNasespecific for RNA in a duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to its template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting as a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein byreference in its entirety, disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic; i.e. new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” (Frohman, 1990),and “one-sided PCR” (Ohara, 1989) which are well-known to those of skillin the art.

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide (Wu andDean, 1996, incorporated herein by reference in its entirety), may alsobe used in the amplification of DNA sequences of the present invention.

Biological Functional Equivalents

Modification and changes may be made in the structure of thepolynucleotides and polypeptides of the present invention and stillobtain a functional molecule that encodes a polypeptide with desirablecharacteristics. As mentioned above, it is often desirable to introduceone or more mutations into a specific polynucleotide sequence. Incertain circumstances, the resulting encoded polypeptide sequence isaltered by this mutation, or in other cases, the sequence of thepolypeptide is unchanged by one or more mutations in the encodingpolynucleotide.

When it is desirable to alter the amino acid sequence of a polypeptideto create an equivalent, or even an improved, second-generationmolecule, the amino acid changes may be achieved by changing one or moreof the codons of the encoding DNA sequence, according to Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines, thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat No. 4,554,101(specifically incorporated herein by reference in its entirety), statesthat the greatest local average hydrophilicity of a protein, as governedby the hydrophilicity of its adjacent amino acids, correlates with abiological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0) threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

In addition, any polynucleotide may be further modified to increasestability in vivo. Possible modifications include, but are not limitedto, the addition of flanking sequences at the 5′ and/or 3′ ends; the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine and wybutosine, as well as acetyl- methyl-,thio- and other modified forms of adenine, cytidine, guanine, thymineand uridine.

In Vivo Polynucleotide Delivery Techniques

In additional embodiments, genetic constructs comprising one or more ofthe polynucleotides of the invention are introduced into cells in vivo.This may be achieved using any of a variety or well known approaches,several of which are outlined below for the purpose of illustration.

1. Adenovirus

One of the preferred methods for in vivo delivery of one or more nucleicacid sequences involves the use of an adenovirus expression vector.“Adenovirus expression vector” is meant to include those constructscontaining adenovirus sequences sufficient to (a) support packaging ofthe construct and (b) to express a polynucleotide that has been clonedtherein in a sense or antisense orientation. Of course, in the contextof an antisense construct, expression does not require that the geneproduct be synthesized.

The expression vector comprises a genetically engineered form of anadenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNA's issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them preferredmRNA's for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kB of DNA. Combined with theapproximately 5.5 kB of DNA that is Ireplaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kB, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-bome cytotoxicity. Also, the replication deficiency ofthe El-deleted virus is incomplete. For example, leakage of viral geneexpression has been observed with the currently available vectors athigh multiplicities of infection (MOT) (Mulligan, 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the currently preferred helper cell line is 293.

Recently, Racher et al. (1995) disclosed improved methods for culturing293 cells and propagating adenovirus. In one format, natural cellaggregates are grown by inoculating individual cells into I litersiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcarriers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and left stationary, with occasional agitation, for1 to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain aconditional replication-defective adenovirus vector for use in thepresent invention, since Adenovirus type 5 is a human adenovirus aboutwhich a great deal of biochemical and genetic information is known, andit has historically been used for most constructions employingadenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the El-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed by Karlsson et al. (1986) or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 109-10ll plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991;

Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993),peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

2. Retroviruses

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding oneor more oligonucleotide or polynucleotide sequences of interest isinserted into the viral genome in the place of certain viral sequencesto produce a virus that is replication-defective. In order to producevirions, a packaging cell line containing the gag, pol, and env genesbut without the LTR and packaging components is constructed (Mann etal., 1983). When a recombinant plasmid containing a cDNA, together withthe retroviral LTR and packaging sequences is introduced into this cellline (by calcium phosphate precipitation for example), the packagingsequence allows the RNA transcript of the recombinant plasmid to bepackaged into viral particles, which are then secreted into the culturemedia (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983).The media containing the recombinant retroviruses is then collected,optionally concentrated, and used for gene transfer. Retroviral vectorsare able to infect a broad variety of cell types. However, integrationand stable expression require the division of host cells (Paskind etal., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

3. Adeno-Associated Viruses

AAV (Ridgeway, 1988; Hermonat and Muzycska, 1984) is a parovirus,discovered as a contamination of adenoviral stocks. It is a ubiquitousvirus (antibodies are present in 85% of the US human population) thathas not been linked to any disease. It is also classified as adependovirus, because its replications is dependent on the presence of ahelper virus, such as adenovirus. Five serotypes have been isolated, ofwhich AAV-2 is the best characterized. AAV has a single-stranded linearDNA that is encapsidated into capsid proteins VP1, VP2 and VP3 to forman icosahedral virion of 20 to 24 nm in diameter (Muzyczka andMcLaughlin, 1988).

The AAV DNA is approximately 4.7 kilobases long. It contains two openreading frames and is flanked by two ITRs. There are two major genes inthe AAV genome: rep and cap. The rep gene codes for proteins responsiblefor viral replications, whereas cap codes for capsid protein VP1-3. EachITR forms a T-shaped hairpin structure. These terminal repeats are theonly essential cis components of the AAV for chromosomal integration.Therefore, the AAV can be used as a vector with all viral codingsequences removed and replaced by the cassette of genes for delivery.Three viral promoters have been identified and named p5, p19, and p40,according to their map position. Transcription from p5 and p19 resultsin production of rep proteins, and transcription from p40 produces thecapsid proteins (Hermonat and Muzyczka, 1984).

There are several factors that prompted researchers to study thepossibility of using rAAV as an expression vector One is that therequirements for delivering a gene to integrate into the host chromosomeare surprisingly few. It is necessary to have the 145-bp ITRs, which areonly 6% of the AAV genome. This leaves room in the vector to assemble a4.5-kb DNA insertion. While this carrying capacity may prevent the AAVfrom delivering large genes, it is amply suited for delivering theantisense constructs of the present invention.

AAV is also a good choice of delivery vehicles due to its safety. Thereis a relatively complicated rescue mechanism: not only wild typeadenovirus but also AAV genes are required to mobilize rAAV. Likewise,AAV is not pathogenic and not associated with any disease. The removalof viral coding sequences minimizes immune reactions to viral geneexpression, and therefore, rAAV does not evoke an inflammatory response.

4. Other Viral Vectors as Expression Constructs

Other viral vectors may be employed as expression constructs in thepresent invention for the delivery of oligonucleotide or polynucleotidesequences to a host cell. Vectors derived from viruses such as vacciniavirus (Ridgeway, 1988; Coupar et al., 1988), lentiviruses, polio virusesand herpes viruses may be employed. They offer several attractivefeatures for various mammalian cells (Friedmann, 1989; Ridgeway, 1988;Coupar et al., 1988; Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. (1991) introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al., 1991).

5. Non-viral Vectors

In order to effect expression of the oligonucleotide or polynucleotidesequences of the present invention, the expression construct must bedelivered into a cell. This delivery may be accomplished in vitro, as inlaboratory procedures for transforming cells lines, or in vivo or exvivo, as in the treatment of certain disease states. As described above,one preferred mechanism for delivery is via viral infection where theexpression construct is encapsulated in an infectious viral particle.

Once the expression construct has been delivered into the cell thenucleic acid encoding the desired oligonucleotide or polynucleotidesequences may be positioned and expressed at different sites. In certainembodiments, the nucleic acid encoding the construct may be stablyintegrated into the genome of the cell. This integration may be in thespecific location and orientation via homologous recombination (genereplacement) or it may be integrated in a random, non-specific location(gene augmentation). In yet further embodiments, the nucleic acid may bestably maintained in the cell as a separate, episomal segment of DNA.Such nucleic acid segments or “episomes” encode sequences sufficient topermit maintenance and replication independent of or in synchronizationwith the host cell cycle. How the expression construct is delivered to acell and where in the cell the nucleic acid remains is dependent on thetype of expression construct employed.

In certain embodiments of the invention, the expression constructcomprising one or more oligonucleotide or polynucleotide sequences maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Reshef (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e. ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

Antisense Oligonucleotides

The end result of the flow of genetic information is the synthesis ofprotein. DNA is transcribed by polymerases into messenger RNA andtranslated on the ribosome to yield a folded, functional protein. Thusthere are several steps along the route where protein synthesis can beinhibited. The native DNA segment coding for a polypeptide describedherein, as all such mammalian DNA strands, has two strands: a sensestrand and an antisense strand held together by hydrogen bonding. Themessenger RNA coding for polypeptide has the same nucleotide sequence asthe sense DNA strand except that the DNA thymidine is replaced byuridine. Thus, synthetic antisense nucleotide sequences will bind to amRNA and inhibit expression of the protein encoded by that mRNA.

The targeting of antisense oligonucleotides to mRNA is thus onemechanism to shut down protein synthesis, and, consequently, representsa powerful and targeted therapeutic approach. For example, the synthesisof polygalactauronase and the muscarine type 2 acetylcholine receptorare inhibited by antisense oligonucleotides directed to their respectivemRNA sequences (U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829,each specifically incorporated herein by reference in its entirety).Further, examples of antisense inhibition have been demonstrated withthe nuclear protein cyclin, the multiple drug resistance gene (MDGI),ICAM−1, E-selectin, STK-1, striatal GABA_(A) receptor and human EGF(Jaskulski et al., 1988; Vasanthakumar and Ahmed, 1989; Peris et al.,1998; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573; U.S. Pat. No.5,718,709 and U.S. Pat. No. 5,610,288, each specifically incorporatedherein by reference in its entirety). Antisense constructs have alsobeen described that inhibit and can be used to treat a variety ofabnormal cellular proliferations, e.g. cancer (U.S. Pat. No. 5,747,470;U.S. Pat. No. 5,591,317 and U.S. Pat. No. 5,783,683, each specificallyincorporated herein by reference in its entirety).

Therefore, in exemplary embodiments, the invention providesoligonucleotide sequences that comprise all, or a portion of, anysequence that is capable of specifically binding to polynucleotidesequence described herein, or a complement thereof. In one embodiment,the antisense oligonucleotides comprise DNA or derivatives thereof.

In another embodiment, the oligonucleotides comprise RNA or derivativesthereof. In a third embodiment, the oligonucleotides are modified DNAscomprising a phosphorothioated modified backbone. In a fourthembodiment, the oligonucleotide sequences comprise peptide nucleic acidsor derivatives thereof. In each case, preferred compositions comprise asequence region that is complementary, and more preferablysubstantially-complementary, and even more preferably, completelycomplementary to one or more portions of polynucleotides disclosedherein.

Selection of antisense compositions specific for a given gene sequenceis based upon analysis of the chosen target sequence (i.e. in theseillustrative examples the rat and human sequences) and determination ofsecondary structure, T_(m), binding energy, relative stability, andantisense compositions were selected based upon their relative inabilityto form dimers, hairpins, or other secondary structures that wouldreduce or prohibit specific binding to the target mRNA in a host cell.

Highly preferred target regions of the mRNA, are those which are at ornear the AUG translation initiation codon, and those sequences whichwere substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationswere performed using v.4 of the OLIGO primer analysis software (Rychlik,1997) and the BLASTN 2.0.5 algorithm software (Altschul et al., 1997).

The use of an antisense delivery method employing a short peptidevector, termed MPG (27 residues), is also contemplated. The MPG peptidecontains a hydrophobic domain derived from the fusion sequence of HIVgp41 and a hydrophilic domain from the nuclear localization sequence ofSV40 T-antigen (Morris et al., 1997). It has been demonstrated thatseveral molecules of the MPG peptide coat the antisense oligonucleotidesand can be delivered into cultured mammalian cells in less than 1 hourwith relatively high efficiency (90%). Further, the interaction with MPGstrongly increases both the stability of the oligonucleotide to nucleaseand the ability to cross the plasma membrane (Morris et al., 1997).

Although proteins traditionally have been used for catalysis of nucleicacids, another class of macromolecules has emerged as useful in thisendeavor. Ribozymes are RNA-protein complexes that cleave nucleic acidsin a site-specific fashion. Ribozymes have specific catalytic domainsthat possess endonuclease activity (Kim and Cech, 1987; Gerlach et al.,1987; Forster and Symons, 1987). For example, a large number ofribozymes accelerate phosphoester transfer reactions with a high degreeof specificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855(specifically incorporated herein by reference) reports that certainribozymes can act as endonucleases with a sequence specificity greaterthan that of known ribonucleases and approaching that of the DNArestriction enzymes. Thus, sequence-specific ribozyme-mediatedinhibition of gene expression may be particularly suited to therapeuticapplications (Scanlon et al., 1991; Sarver et al., 1990). Recently, itwas reported that ribozymes elicited genetic changes in some cells linesto which they were applied; the altered genes included the oncogenesH-ras, c-fos and genes of HIV. Most of this work involved themodification of a target mRNA, based on a specific mutant codon that iscleaved by a specific ribozyme.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofa enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., 1992). Thus, thespecificity of action of a ribozyme is greater than that of an antisenseoligonucleotide binding the same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis 8 virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. (1992).Examples of hairpin motifs are described by Hampel et al. (Eur. Pat.Appl. Publ. No. EP 0360257), Hampel and Tritz (1989), Hampel et al.(1990) and U.S. Pat. No. 5,631,359 (specifically incorporated herein byreference). An example of the hepatitis 8 virus motif is described byPerrotta and Been (1992); an example of the RNaseP motif is described byGuerrier-Takada et al. (1983); Neurospora VS RNA ribozyme motif isdescribed by Collins (Saville and Collins, 1990; Saville and Collins,1991; Collins and Olive, 1993); and an example of the Group I intron isdescribed in (U.S. Pat. No. 4,987,071, specifically incorporated hereinby reference). All that is important in an enzymatic nucleic acidmolecule of this invention is that it has a specific substrate bindingsite which is complementary to one or more of the target gene RNAregions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

In certain embodiments, it may be important to produce enzymaticcleaving agents which exhibit a high degree of specificity for the RNAof a desired target, such as one of the sequences disclosed herein. Theenzymatic nucleic acid molecule is preferably targeted to a highlyconserved sequence region of a target mRNA. Such enzymatic nucleic acidmolecules can be delivered exogenously to specific cells as required.Alternatively, the ribozymes can be expressed from DNA or RNA vectorsthat are delivered to specific cells.

Small enzymatic nucleic acid motifs (e.g., of the hammerhead or thehairpin structure) may also be used for exogenous delivery. The simplestructure of these molecules increases the ability of the enzymaticnucleic acid to invade targeted regions of the mRNA structure.Alternatively, catalytic RNA molecules can be expressed within cellsfrom eukaryotic promoters (e.g., Scanlon et al., 1991; Kashani-Sabet etal., 1992; Dropulic et al., 1992; Weerasinghe et al., 1991; Ojwang etal., 1992; Chen et al., 1992; Sarver et al., 1990). Those skilled in theart realize that any ribozyme can be expressed in eukaryotic cells fromthe appropriate DNA vector. The activity of such ribozymes can beaugmented by their release from the primary transcript by a secondribozyme (Int. Pat. Appl. Publ. No. WO 93/23569, and Int. Pat. Appl.Publ. No. WO 94/02595, both hereby incorporated by reference; Ohkawa etal., 1992; Taira et al., 1991; and Ventura et al., 1993).

Ribozymes may be added directly, or can be complexed with cationiclipids, lipid complexes, packaged within liposomes, or otherwisedelivered to target cells. The RNA or RNA complexes can be locallyadministered to relevant tissues ex vivo, or in vivo through injection,aerosol inhalation, infusion pump or stent, with or without theirincorporation in biopolymers.

Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specificallyincorporated herein by reference) and synthesized to be tested in vitroand in vivo, as described. Such ribozymes can also be optimized fordelivery. While specific examples are provided, those in the art willrecognize that equivalent RNA targets in other species can be utilizedwhen necessary.

Hammerhead or hairpin ribozymes may be individually analyzed by computerfolding (Jaeger et al., 1989) to assess whether the ribozyme sequencesfold into the appropriate secondary structure. Those ribozymes withunfavorable intramolecular interactions between the binding arms and thecatalytic core are eliminated from consideration. Varying binding armlengths can be chosen to optimize activity. Generally, at least 5 or sobases on each arm are able to bind to, or otherwise interact with, thetarget RNA.

Ribozymes of the hammerhead or hairpin motif may be designed to annealto various sites in the mRNA message, and can be chemically synthesized.The method of synthesis used follows the procedure for normal RNAsynthesis as described in Usman et al. (1987) and in Scaringe etal.(1990) and makes use of common nucleic acid protecting and couplinggroups, such as dimethoxytrityl at the 5′-end, and phosphoramidites atthe 3′-end. Average stepwise coupling yields are typically >98%. Hairpinribozymes may be synthesized in two parts and annealed to reconstruct anactive ribozyme (Chowrira and Burke, 1992). Ribozymes may be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-o-methyl, 2′-H(for a review see e.g., Usman and Cedergren, 1992). Ribozymes may bepurified by gel electrophoresis using general methods or by highpressure liquid chromatography and resuspended in water.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al, 1990;Pieken et al., 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ.No. WO 93/15187; Int. Pat. Appl. Pubi. No. WO 91/03162; Eur. Pat. Appl.Pubi. No.92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes thegeneral methods for delivery of enzymatic RNA molecules. Ribozymes maybe administered to cells by a variety of methods known to those familiarto the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl.Publ. No. WO 93/23569, each specifically incorporated herein byreference.

Another means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol II or pol III promoters will be expressed at high levels in allcells; the levels of a given pol II promoter in a given cell type willdepend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersmay also be used, providing that the prokaryotic RNA polymerase enzymeis expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gaoand Huang, 1993; Lieber et al., 1993; Zhou et al., 1990). Ribozymesexpressed from such promoters can function in mammalian cells (e.g.Kashani-Saber et al., 1992; Ojwang et al., 1992; Chen et al., 1992; Yuet al., 1993; L'Huillier et al., 1992; Lisziewicz et al., 1993). Suchtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated vectors), or viral RNA vectors (such as retroviral,semliki forest virus, sindbis virus vectors).

Ribozymes may be used as diagnostic tools to examine genetic drift andmutations within diseased cells. They can also be used to assess levelsof the target RNA molecule. The close relationship between ribozymeactivity and the structure of the target RNA allows the detection ofmutations in any region of the molecule which alters the base-pairingand three-dimensional structure of the target RNA. By using multipleribozymes, one may map nucleotide changes which are important to RNAstructure and function in vitro, as well as in cells and tissues.Cleavage of target RNAs with ribozymes may be used to inhibit geneexpression and define the role (essentially) of specified gene productsin the progression of disease. In this manner, other genetic targets maybe defined as important mediators of the disease. These studies willlead to better treatment of the disease progression by affording thepossibility of combinational therapies (e.g., multiple ribozymestargeted to different genes, ribozymes coupled with known small moleculeinhibitors, or intermittent treatment with combinations of ribozymesand/or other chemical or biological molecules). Other in vitro uses ofribozymes are well known in the art, and include detection of thepresence of mRNA associated with an IL-5 related condition. Such RNA isdetected by determining the presence of a cleavage product aftertreatment with a ribozyme using standard methodology.

Peptide Nucleic Acids

In certain embodiments, the inventors contemplate the use of peptidenucleic acids (PNAs) in the practice of the methods of the invention.PNA is a DNA mimic in which the nucleobases are attached to apseudopeptide backbone (Good and Nielsen, 1997). PNA is able to beutilized in a number methods that traditionally have used RNA or DNA.Often PNA sequences perform better in techniques than the correspondingRNA or DNA sequences and have utilities that are not inherent to RNA orDNA. A review of PNA including methods of making, characteristics of,and methods of using, is provided by Corey (1997) and is incorporatedherein by reference. As such, in certain embodiments, one may preparePNA sequences that are complementary to one or more portions of the ACEmRNA sequence, and such PNA compositions may be used to regulate, alter,decrease, or reduce the translation of ACE-specific mRNA, and therebyalter the level of ACE activity in a host cell to which such PNAcompositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normalphosphodiester backbone of DNA (Nielsen et al., 1991; Hanvey et al.,1992; Hyrup and Nielsen, 1996; Neilsen, 1996). This chemistry has threeimportant consequences: firstly, in contrast to DNA or phosphorothioateoligonucleotides, PNAs are neutral molecules; secondly, PNAs areachiral, which avoids the need to develop a stereoselective synthesis;and thirdly, PNA synthesis uses standard Boc (Dueholm et al., 1994) orFmoc (Thomson et al., 1995) protocols for solid-phase peptide synthesis,although other methods, including a modified Merrifield method, havebeen used (Christensen et al., 1995).

PNA monomers or ready-made oligomers are commercially available fromPerSeptive Biosystems (Framingham, MA). PNA syntheses by either Boc orFmoc protocols are straightforward using manual or automated protocols(Norton et al., 1995). The manual protocol lends itself to theproduction of chemically modified PNAs or the simultaneous synthesis offamilies of closely related PNAs.

As with peptide synthesis, the success of a particular PNA synthesiswill depend on the properties of the chosen sequence. For example, whilein theory PNAs can incorporate any combination of nucleotide bases, thepresence of adjacent purines can lead to deletions of one or moreresidues in the product. In expectation of this difficulty, it issuggested that, in producing PNAs with adjacent purines, one shouldrepeat the coupling of residues likely to be added inefficiently. Thisshould be followed by the purification of PNAs by reverse-phasehigh-pressure liquid chromatography (Norton et al., 1995) providingyields and purity of product similar to those observed during thesynthesis of peptides.

Modifications of PNAs for a given application may be accomplished bycoupling amino acids during solid-phase synthesis or by attachingcompounds that contain a carboxylic acid group to the exposedN-terrninal amine. Alternatively, PNAs can be modified after synthesisby coupling to an introduced lysine or cysteine. The ease with whichPNAs can be modified facilitates optimization for better solubility orfor specific functional requirements. Once synthesized, the identity ofPNAs and their derivatives can be confirmed by mass spectrometry.Several studies have made and utilized modifications of PNAs (Norton etal., 1995; Haaima et al., 1996; Stetsenko et al., 1996; Petersen et al.,1995; Ulmann et al., 1996; Koch et al., 1995; Orum et al., 1995; Footeret al., 1996; Griffith et al., 1995; Kremsky et al., 1996; Pardridge etal., 1995; Boffa et al., 1995; Landsdorp et al., 1996;Gambacorti-Passerini et al., 1996; Armitage et al., 1997; Seeger et al.,1997; Ruskowski et al., 1997). U.S. Pat. No. 5,700,922 discussesPNA-DNA-PNA chimeric molecules and their uses in diagnostics, modulatingprotein in organisms, and treatment of conditions susceptible totherapeutics.

In contrast to DNA and RNA, which contain negatively charged linkages,the PNA backbone is neutral. In spite of this dramatic alteration, PNAsrecognize complementary DNA and RNA by Watson-Crick pairing (Egholm etal., 1993), validating the initial modeling by Nielsen et al. (1991).PNAs lack 3′ to 5′ polarity and can bind in either parallel orantiparallel fashion, with the antiparallel mode being preferred (Egholmet al., 1993).

Hybridization of DNA oligonucleotides to DNA and RNA is destabilized byelectrostatic repulsion between the negatively charged phosphatebackbones of the complementary strands. By contrast, the absence ofcharge repulsion in PNA-DNA or PNA-RNA duplexes increases the meltingtemperature (T_(m)) and reduces the dependence of T_(m) on theconcentration of mono- or divalent cations (Nielsen et al., 1991). Theenhanced rate and affinity of hybridization are significant because theyare responsible for the surprising ability of PNAs to perform strandinvasion of complementary sequences within relaxed double-stranded DNA.In addition, the efficient hybridization at inverted repeats suggeststhat PNAs can recognize secondary structure effectively withindouble-stranded DNA. Enhanced recognition also occurs with PNAsimmobilized on surfaces, and Wang et al. have shown that support-boundPNAs can be used to detect hybridization events (Wang et al., 1996).

One might expect that tight binding of PNAs to complementary sequenceswould also increase binding to similar (but not identical) sequences,reducing the sequence specificity of PNA recognition. As with DNAhybridization, however, selective recognition can be achieved bybalancing oligomer length and incubation temperature. Moreover,selective hybridization of PNAs is encouraged by PNA-DNA hybridizationbeing less tolerant of base mismatches than DNA-DNA hybridization. Forexample, a single mismatch within a 16 bp PNA-DNA duplex can reduce theT_(m) by up to 15° C. (Egholm et al., 1993). This high level ofdiscrimination has allowed the development of several PNA-basedstrategies for the analysis of point mutations (Wang et al., 1996;Carlsson et al., 1996; Thiede et al., 1996; Webb and Hurskainen, 1996;Perry-O'Keefe et al., 1996).

High-affinity binding provides clear advantages for molecularrecognition and the development of new applications for PNAs. Forexample, 1-13 nucleotide PNAs inhibit the activity of telomerase, aribonucleo-protein that extends telomere ends using an essential RNAtemplate, while the analogous DNA oligomers do not (Norton et al.,1996).

Neutral PNAs are more hydrophobic than analogous DNA oligomers, and thiscan lead to difficulty solubilizing them at neutral pH, especially ifthe PNAs have a high purine content or if they have the potential toform secondary structures. Their solubility can be enhanced by attachingone or more positive charges to the PNA termini (Nielsen et al., 1991).

Findings by Allfrey and colleagues suggest that strand invasion willoccur spontaneously at sequences within chromosomal DNA (Boffa et al.,1995; Boffa et al., 1996). These studies targeted PNAs to tripletrepeats of the nucleotides CAG and used this recognition to purifytranscriptionally active DNA (Boffa et al., 1995) and to inhibittranscription (Boffa et al., 1996). This result suggests that if PNAscan be delivered within cells then they will have the potential to begeneral sequence-specific regulators of gene expression. Studies andreviews concerning the use of PNAs as antisense and anti-gene agentsinclude Nielsen et al. (1993b), Hanvey et al. (1992), and Good andNielsen (1997). Koppelhus et al. (1997) have used PNAs to inhibit HIV-Iinverse transcription, showing that PNAs may be used for antiviraltherapies.

Methods of characterizing the antisense binding properties of PNAs arediscussed in Rose (1993) and Jensen et al. (1997). Rose uses capillarygel electrophoresis to determine binding of PNAs to their complementaryoligonucleotide, measuring the relative binding kinetics andstoichiometry. Similar types of measurements were made by Jensen et al.using BIAcore™ technology.

Other applications of PNAs include use in DNA strand invasion (Nielsenet al., 1991), antisense inhibition (Hanvey et al., 1992), mutationalanalysis (Orum et al., 1993), enhancers of transcription (Mollegaard etal., 1994), nucleic acid purification (Orum et al., 1995), isolation oftranscriptionally active genes (Boffa et al., 1995), blocking oftranscription factor binding (Vickers et al., 1995), genome cleavage(Veselkov et al., 1996), biosensors (Wang et al., 1996), in situhybridization (Thisted et al., 1996), and in a alternative to Southernblotting (Perry-O'Keefe, 1996).

Polypeptide Compositions

The present invention, in other aspects, provides polypeptidecompositions. Generally, a polypeptide of the invention will be anisolated polypeptide (or an epitope, variant, or active fragmentthereof) derived from a mammalian species. Preferably, the polypeptideis encoded by a polynucleotide sequence disclosed herein or a sequencewhich hybridizes under moderately stringent conditions to apolynucleotide sequence disclosed herein. Alternatively, the polypeptidemay be defined as a polypeptide which comprises a contiguous amino acidsequence from an amino acid sequence disclosed herein, or whichpolypeptide comprises an entire amino acid sequence disclosed herein.

In the present invention, a polypeptide composition is also understoodto comprise one or more polypeptides that are immunologically reactivewith antibodies generated against a polypeptide of the invention,particularly a polypeptide having the amino acid sequence disclosed inSEQ ID NO: 112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380,383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551,553-568, 573-586, 588-590, 592, 706-708, 775, 776, 778 and 780, oractive fragments, variants or biological functional equivalents thereof.

Likewise, a polypeptide composition of the present invention isunderstood to comprise one or more polypeptides that are capable ofeliciting antibodies that are immunologically reactive with one or morepolypeptides encoded by one or more contiguous nucleic acid sequencescontained in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315,326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530,531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774,777, 789, 817, 823 and 824, or to active fragments, or to variantsthereof, or to one or more nucleic acid sequences which hybridize to oneor more of these sequences under conditions of moderate to highstringency. Particularly illustrative polypeptides include the aminoacid sequence disclosed in SEQ ID NO: 112-114, 172, 176, 178, 327, 329,331, 336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525,527, 532, 534, 537-551, 553-568, 573-586, 588-590, 592, 706-708, 775,776, 778 and 780.

As used herein, an active fragment of a polypeptide includes a whole ora portion of a polypeptide which is modified by conventional techniques,e.g., mutagenesis, or by addition, deletion, or substitution, but whichactive fragment exhibits substantially the same structure function,antigenicity, etc., as a polypeptide as described herein.

In certain illustrative embodiments, the polypeptides of the inventionwill comprise at least an immunogenic portion of a prostate-specificprotein or a variant thereof, as described herein. As noted above, a“prostate-specific protein” is a protein that is expressed by prostatecells. Proteins that are prostate-specific proteins also reactdetectably within an immunoassay (such as an ELISA) with antisera from apatient with prostate cancer. Polypeptides as described herein may be ofany length. Additional sequences derived from the native protein and/orheterologous sequences may be present, and such sequences may (but neednot) possess further immunogenic or antigenic properties.

An “immunogenic portion,” as used herein is a portion of a protein thatis recognized (i.e., specifically bound) by a B-cell and/or T-cellsurface antigen receptor. Such immunogenic portions generally compriseat least 5 amino acid residues, more preferably at least 10, and stillmore preferably at least 20 amino acid residues of a prostate-specificprotein or a variant thereof. Certain preferred immunogenic portionsinclude peptides in which an N-terminal leader sequence and/ortransmembrane domain have been deleted. Other preferred immunogenicportions may contain a small N- and/or C-terminal deletion (e.g., 1-30amino acids, preferably 5-15 amino acids), relative to the matureprotein.

Immunogenic portions may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Suchtechniques include screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well known techniques. An immunogenic portion of anative prostate-specific protein is a portion that reacts with suchantisera and/or T-cells at a level that is not substantially less thanthe reactivity of the full length polypeptide (e.g., in an ELISA and/orT-cell reactivity assay). Such immunogenic portions may react withinsuch assays at a level that is similar to or greater than the reactivityof the full length polypeptide. Such screens may generally be performedusing methods well known to those of ordinary skill in the art, such asthose described in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. For example, a polypeptide may beimmobilized on a solid support and contacted with patient sera to allowbinding of antibodies within the sera to the immobilized polypeptide.Unbound sera may then be removed and bound antibodies detected using,for example, ¹²⁵I-labeled Protein A.

As noted above, a composition may comprise a variant of a nativeprostate-specific protein. A polypeptide “variant,” as used herein, is apolypeptide that differs from a native prostate-specific protein in oneor more substitutions, deletions, additions and/or insertions, such thatthe immunogenicity of the polypeptide is not substantially diminished.In other words, the ability of a variant to react with antigen-specificantisera may be enhanced or unchanged, relative to the native protein,or may be diminished by less than 50%, and preferably less than 20%,relative to the native protein. Such variants may generally beidentified by modifying one of the above polypeptide sequences andevaluating the reactivity of the modified polypeptide withantigen-specific antibodies or antisera as described herein. Preferredvariants include those in which one or more portions, such as anN-terminal leader sequence or transmembrane domain, have been removed.Other preferred variants include variants in which a small portion(e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removedfrom the N- and/or C-terminal of the mature protein.

Polypeptide variants encompassed by the present invention include thoseexhibiting at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% or more identity (determined as describedabove) to the polypeptides disclosed herein.

Preferably, a variant contains conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. Amino acid substitutions may generally be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. In a preferredembodiment, variant polypeptides differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer.Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theimmunogenicity, secondary structure and hydropathic nature of thepolypeptide.

As noted above, polypeptides may comprise a signal (or leader) sequenceat the N-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

Polypeptides may be prepared using any of a variety of well knowntechniques. Recombinant polypeptides encoded by DNA sequences asdescribed above may be readily prepared from the DNA sequences using anyof a variety of expression vectors known to those of ordinary skill inthe art. Expression may be achieved in any appropriate host cell thathas been transformed or transfected with an expression vector containinga DNA molecule that encodes a recombinant polypeptide. Suitable hostcells include prokaryotes, yeast, and higher eukaryotic cells, such asmammalian cells and plant cells. Preferably, the host cells employed areE. coli, yeast or a mammalian cell line such as COS or CHO. Supernatantsfrom suitable host/vector systems which secrete recombinant protein orpolypeptide into culture media may be first concentrated using acommercially available filter. Following concentration, the concentratemay be applied to a suitable purification matrix such as an affinitymatrix or an ion exchange resin. Finally, one or more reverse phase HPLCsteps can be employed to further purify a recombinant polypeptide.

Portions and other variants having less than about 100 amino acids, andgenerally less than about 50 amino acids, may also be generated bysynthetic means, using techniques well known to those of ordinary skillin the art. For example, such polypeptides may be synthesized using anyof the commercially available solid-phase techniques, such as theMerrifield solid-phase synthesis method, where amino acids aresequentially added to a growing amino acid chain. See Merrifield, J. Am.Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City, Calif.), and may beoperated according to the manufacturer's instructions.

Within certain specific embodiments, a polypeptide may be a fusionprotein that comprises multiple polypeptides as described herein, orthat comprises at least one Ipolypeptide as described herein and anunrelated sequence, such as a known tumor protein. A fusion partner may,for example, assist in providing T helper epitopes (an immunologicalfusion partner), preferably T helper epitopes recognized by humans, ormay assist in expressing the protein (an expression enhancer) at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the protein or to enable the protein to be targeted todesired intracellular compartments. Still further fusion partnersinclude affinity tags, which facilitate purification of the protein.

Fusion proteins may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion protein isexpressed as a recombinant protein, allowing the production of increasedlevels, relative to a non-fused protein, in an expression system.Briefly, DNA sequences encoding the polypeptide components may beassembled separately, and ligated into an appropriate expression vector.The 3′ end of the DNA sequence encoding one polypeptide component isligated, with or without a peptide linker, to the 5′ end of a DNAsequence encoding the second polypeptide component so that the readingframes of the sequences are in phase. This permits translation into asingle fusion protein that retains the biological activity of bothcomponent polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may generally be from 1 to about 50 amino acids inlength. Linker sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

Fusion proteins are also provided. Such proteins comprise a polypeptideas described herein together with an unrelated immunogenic protein.Preferably the immunogenic protein is capable of eliciting a recallresponse. Examples of such proteins include tetanus, tuberculosis andhepatitis proteins (see, for example, Stoute et al. New Engl. J. Med.,336:86-91, 1997).

Within preferred embodiments, an immunological fusion partner is derivedfrom protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292, 1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionprotein. A repeat portion is found in the C-terminal region starting atresidue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

In general, polypeptides (including fusion proteins) and polynucleotidesas described herein are isolated. An “isolated” polypeptide orpolynucleotide is one that is removed from its original environment. Forexample, a naturally-occurring protein is isolated if it is separatedfrom some or all of the coexisting materials in the natural system.Preferably, such polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not a part of the naturalenvironment.

Binding Agents

The present invention further provides agents, such as antibodies andantigen-binding fragments thereof, that specifically bind to aprostate-specific protein. As used herein, an antibody, orantigen-binding fragment thereof, is said to “specifically bind” to aprostate-specific protein if it reacts at a detectable level (within,for example, an ELISA) with a prostate-specific protein, and does notreact detectably with unrelated proteins under similar conditions. Asused herein, “binding” refers to a noncovalent association between twoseparate molecules such that a complex is formed. The ability to bindmay be evaluated by, for example, determining a binding constant for theformation of the complex. The binding constant is the value obtainedwhen the concentration of the complex is divided by the product of thecomponent concentrations. In general, two compounds are said to “bind,”in the context of the present invention, when the binding constant forcomplex formation exceeds about 10³ L/mol. The binding constant may bedetermined using methods well known in the art.

Binding agents may be further capable of differentiating betweenpatients with and without a cancer, such as prostate cancer, using therepresentative assays provided herein. In other words, antibodies orother binding agents that bind to a prostate-specific protein willgenerate a signal indicating the presence of a cancer in at least about20% of patients with the disease, and will generate a negative signalindicating the absence of the disease in at least about 90% ofindividuals without the cancer. To determine whether a binding agentsatisfies this requirement, biological samples (e.g., blood, sera,sputum, urine and/or tumor biopsies) from patients with and without acancer (as determined using standard clinical tests) may be assayed asdescribed herein for the presence of polypeptides that bind to thebinding agent. It will be apparent that a statistically significantnumber of samples with and without the disease should be assayed. Eachbinding agent should satisfy the above criteria; however, those ofordinary skill in the art will recognize that binding agents may be usedin combination to improve sensitivity.

Any agent that satisfies the above requirements may be a binding agent.For example, a binding agent may be a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, antibodies can be produced by cell culturetechniques, including the generation of monoclonal antibodies asdescribed herein, or via transfection of antibody genes into suitablebacterial or mammalian cell hosts, in order to allow for the productionof recombinant antibodies. In one technique, an immunogen comprising thepolypeptide is initially injected into any of a wide-variety of mammals(e.g., mice, rats, rabbits, sheep or goats). In this step, thepolypeptides of this invention may serve as the immunogen withoutImodification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for an antigenic polypeptide of interestmay be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses FIAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

Within certain embodiments, the use of antigen-binding fragments ofantibodies may be preferred. Such fragments include Fab fragments, whichmay be prepared using standard techniques. Briefly, immunoglobulins maybe purified from rabbit serum by affinity chromatography on Protein Abead columns (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988) and digested by papain to yield Fab andFc fragments. The Fab and Fc fragments may be separated by affinitychromatography on protein A bead columns.

Monoclonal antibodies of the present invention may be coupled to one ormore therapeutic agents. Suitable agents in this regard includeradionuclides, differentiation inducers, drugs, toxins, and derivativesthereof. Preferred radionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re,¹⁸⁸Re, ²¹¹At, and ²¹²Bi. Preferred drugs include methotrexate, andpyrimidine and purine analogs. Preferred differentiation inducersinclude phorbol esters and butyric acid. Preferred toxins include ricin,abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin,Shigella toxin, and pokeweed antiviral protein.

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino orsulffiydryl group, on one may be capable of reacting with acarbonyl-containing group, such as an anhydride or an acid halide, orwith an alkyl group containing a good leaving group (e.g., a halide) onthe other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used. Alternatively, a carrier can be used.

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group. Suitable carriers includeproteins such as albumins (e.g., U.S. Pat. No. 4,507,234, to Kato etal.), peptides and polysaccharides such as aminodextran (e.g., U.S. Pat.No. 4,699,784, to Shih et al.). A carrier may also bear an agent bynoncovalent bonding or by encapsulation, such as within a liposomevesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriersspecific for radionuclide agents include radiohalogenated smallmolecules and chelating compounds. For example, U.S. Pat. No. 4,735,792discloses representative radiohalogenated small molecules and theirsynthesis. A radionuclide chelate may be formed from chelating compoundsthat include those containing nitrogen and sulfur atoms as the donoratoms for binding the metal, or metal oxide, radionuclide. For example,U.S. Pat. No. 4,673,562, to Davison et al. discloses representativechelating compounds and their synthesis.

A variety of routes of administration for the antibodies andimmunoconjugates may be used. Typically, administration will beintravenous, intramuscular, subcutaneous or in the bed of a resectedtumor. It will be evident that the precise dose of theantibody/immunoconjugate will vary depending upon the antibody used, theantigen density on the tumor, and the rate of clearance of the antibody.

T Cells

Immunotherapeutic compositions may also, or alternatively, comprise Tcells specific for a prostate-specific protein. Such cells may generallybe prepared in vitro or ex vivo, using standard procedures. For example,T cells may be isolated from bone marrow, peripheral blood, or afraction of bone marrow or peripheral blood of a patient, using acommercially available cell separation system, such as the Isolex™System, available from Nexell Therapeutics, Inc. (Irvine, Calif.; seealso U.S. Pat. No. 5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO91/16116 and WO 92/07243). Alternatively, T cells may be derived fromrelated or unrelated humans, non-human mammals, cell lines or cultures.

T cells may be stimulated with a prostate-specific polypeptide,polynucleotide encoding a prostate-specific polypeptide and/or anantigen presenting cell (APC) that expresses such a polypeptide. Suchstimulation is performed under conditions and for a time sufficient topermit the generation of T cells that are specific for the polypeptide.Preferably, a prostate-specific polypeptide or polynucleotide is presentwithin a delivery vehicle, such as a microsphere, to facilitate thegeneration of specific T cells.

T cells are considered to be specific for a prostate-specificpolypeptide if the T cells specifically proliferate, secrete cytokinesor kill target cells coated with the polypeptide or expressing a geneencoding the polypeptide. T cell specificity may be evaluated using anyof a variety of standard techniques. For example, within a chromiumrelease assay or proliferation assay, a stimulation index of more thantwo fold increase in lysis and/or proliferation, compared to negativecontrols, indicates T cell specificity. Such assays may be performed,for example, as described in Chen et al., Cancer Res. 54:1065-1070,1994. Alternatively, detection of the proliferation of T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring an increased rate of DNAsynthesis (e.g., by pulse-labeling cultures of T cells with tritiatedthymidine and measuring the amount of tritiated thymidine incorporatedinto DNA). Contact with a prostate-specific polypeptide (100 ng/ml-100μg/ml, preferably 200 ng/ml-25 μg/ml) for 3-7 days should result in atleast a two fold increase in proliferation of the T cells. Contact asdescribed above for 2-3 hours should result in activation of the Tcells, as measured using standard cytokine assays in which a two foldincrease in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1, Wiley Interscience (Greene 1998)). T cells thathave been activated in response to a prostate-specific polypeptide,polynucleotide or polypeptide-expressing APC may be CD4⁺ and/or CD8⁺.prostate-specific protein-specific T cells may be expanded usingstandard techniques. Within preferred embodiments, the T cells arederived from a patient, a related donor or an unrelated donor, and areadministered to the patient following stimulation and expansion.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate inresponse to a prostate-specific polypeptide, polynucleotide or APC canbe expanded in number either in vitro or in vivo. Proliferation of suchT cells in vitro may be accomplished in a variety of ways. For example,the T cells can be re-exposed to a prostate-specific polypeptide, or ashort peptide corresponding to an immunogenic portion of such apolypeptide, with or without the addition of T cell growth factors, suchas interleukin-2, and/or stimulator cells that synthesize aprostate-specific polypeptide. Alternatively, one or more T cells thatproliferate in the presence of a prostate-specific protein can beexpanded in number by cloning. Methods for cloning cells are well knownin the art, and include limiting dilution.

Pharmaceutical Compositions

In additional embodiments, the present invention concerns formulation ofone or more of the polynucleotide, polypeptide, T-cell and/or antibodycompositions disclosed herein in pharmaceutically-acceptable solutionsfor administration to a cell or an animal, either alone, or incombination with one or more other modalities of therapy.

It will also be understood that, if desired, the nucleic acid segment,RNA, DNA or PNA compositions that express a polypeptide as disclosedherein may be administered in combination with other agents as well,such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The compositions may thus be deliveredalong with various other agents as required in the particular instance.Such compositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

1. Oral Delivery

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al.,1997; Hwang et al., 1998; U.S. Pat. No. 5,641,515; U.S. Pat. No.5,580,579 and U.S. Pat. No. 5,792,451, each specifically incorporatedherein by reference in its entirety). The tablets, troches, pills,capsules and the like may also contain the following: a binder, as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.A syrup of elixir may contain the active compound sucrose as asweetening agent methyl and propylparabens as preservatives, a dye andflavoring, such as cherry or orange flavor. Of course, any material usedin preparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

2. Injectable Delivery

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally as describedin U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No.5,399,363 (each specifically incorporated herein by reference in itsentirety). Solutions of the active compounds as free base orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug-release capsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

3. Nasal Delivery

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, nucleic acids, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212(each specifically incorporated herein by reference in its entirety).Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S.Pat. No. 5,725,871, specifically incorporated herein by reference in itsentirety) are also well-known in the pharmaceutical arts. Likewise,transmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045 (specificallyincorporated herein by reference in its entirety).

4. Liposome-, Nanocapsule-, and Microparticle-Mediated Delivery

In certain embodiments, the inventors contemplate the use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, or ananoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically-acceptable formulations of the nucleic acids orconstructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art (see for example, Couvreuret al., 1977; Couvreur, 1988; Lasic, 1998; which describes the use ofliposomes and nanocapsules in the targeted antibiotic therapy forintracellular bacterial infections and diseases). Recently, liposomeswere developed with improved serum stability and circulation half-times(Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987; U.S. Pat. No.5,741,516, specifically incorporated herein by reference in itsentirety). Further, various methods of liposome and liposome likepreparations as potential drug carriers have been reviewed (Takakura,1998; Chandran et al., 1997; Margalit, 1995; U.S. Pat. No. 5,567,434;U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No.5,738,868 and U.S. Pat. No. 5,795,587, each specifically incorporatedherein by reference in its entirety).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., 1990; Muller et al., 1990). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes, drugs(Heath and Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989;Fresta and Puglisi, 1996), radiotherapeutic agents (Pikul et al., 1987),enzymes (Imaizumi et al., 1990a; Imaizumi et al., 1990b), viruses(Faller and Baltimore, 1984), transcription factors and allostericeffectors (Nicolau and Gersonde, 1979) into a variety of cultured celllines and animals. In addition, several successful clinical trailsexamining the effectiveness of liposome-mediated drug delivery have beencompleted (Lopez-Berestein et al., 1985a; 1985b; Coune, 1988; Sculier etal., 1988). Furthermore, several studies suggest that the use ofliposomes is not associated with autoimmune responses, toxicity orgonadal localization after systemic delivery (Mori and Fukatsu, 1992).

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Liposomes bear resemblance to cellular membranes and are contemplatedfor use in connection with the present invention as carriers for thepeptide compositions. They are widely suitable as both water- andlipid-soluble substances can be entrapped, i.e. in the aqueous spacesand within the bilayer itself, respectively. It is possible that thedrug-bearing liposomes may even be employed for site-specific deliveryof active agents by selectively modifying the liposomal formulation.

In addition to the teachings of Couvreur et al. (1977; 1988), thefollowing information may be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

In addition to temperature, exposure to proteins can alter thepermeability of liposomes. Certain soluble proteins, such as cytochromec, bind, deform and penetrate the bilayer, thereby causing changes inpermeability. Cholesterol inhibits this penetration of proteins,apparently by packing the phospholipids more tightly. It is contemplatedthat the most useful liposome formations for antibiotic and inhibitordelivery will contain cholesterol.

The ability to trap solutes varies between different types of liposomes.For example, MLVs are moderately efficient at trapping solutes, but SUVsare extremely inefficient. SUVs offer the advantage of homogeneity andreproducibility in size distribution, however, and a compromise betweensize and trapping efficiency is offered by large unilamellar vesicles(LUVs). These are prepared by ether evaporation and are three to fourtimes more efficient at solute entrapment than MLVs.

In addition to liposome characteristics, an important determinant inentrapping compounds is the physicochemical properties of the compounditself. Polar compounds are trapped in the aqueous spaces and nonpolarcompounds bind to the lipid bilayer of the vesicle. Polar compounds arereleased through permeation or when the bilayer is broken, but nonpolarcompounds remain affiliated with the bilayer unless it is disrupted bytemperature or exposure to lipoproteins. Both types show maximum effluxrates at the phase transition temperature.

Liposomes interact with cells via four different mechanisms: endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. It often is difficult to determine which mechanism isoperative and more than one may operate at the same time.

The fate and disposition of intravenously injected liposomes depend ontheir physical properties, such as size, fluidity, and surface charge.They may persist in tissues for h or days, depending on theircomposition, and half lives in the blood range from min to several h.Larger liposomes, such as MLVs and LUVs, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominate site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehavior limits the potential targeting of liposomes to only thoseorgans and tissues accessible to their large size. These include theblood, liver, spleen, bone marrow, and lymphoid organs.

Targeting is generally not a limitation in terms of the presentinvention. However, should specific targeting be desired, methods areavailable for this to be accomplished. Antibodies may be used to bind tothe liposome surface and to direct the antibody and its drug contents tospecific antigenic receptors located on a particular cell-type surface.Carbohydrate determinants (glycoprotein or glycolipid cell-surfacecomponents that play a role in cell-cell recognition, interaction andadhesion) may also be used as recognition sites as they have potentialin directing liposomes to particular cell types. Mostly, it iscontemplated that intravenous injection of liposomal preparations wouldbe used, but other routes of administration are also conceivable.

Alternatively, the invention provides for pharmaceutically-acceptablenanocapsule formulations of the compositions of the present invention.Nanocapsules can generally entrap compounds in a stable and reproducibleway (Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998;Douglas et al., 1987). To avoid side effects due to intracellularpolymeric overloading, such ultrafine particles (sized around 0.1 μm)should be designed using polymers able to be degraded in vivo.Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet theserequirements are contemplated for use in the present invention. Suchparticles may be are easily made, as described (Couvreur et al., 1980;1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry etal., 1995 and U.S. Pat. No. 5,145,684, specifically incorporated hereinby reference in its entirety).

Immunogenic Compositions

In certain preferred embodiments of the present invention, immunogeniccompositions, or vaccines, are provided. The immunogenic compositionswill generally comprise one or more pharmaceutical compositions, such asthose discussed above, in combination with an immunostimulant. Animmunostimulant may be any substance that enhances or potentiates animmune response (antibody and/or cell-mediated) to an exogenous antigen.Examples of immunostimulants include adjuvants, biodegradablemicrospheres (e.g., polylactic galactide) and liposomes (into which thecompound is incorporated; see e.g., Fullerton, U.S. Pat. No. 4,235,877).Vaccine preparation is generally described in, for example, M. F. Powelland M. J. Newman, eds., “Vaccine Design (the subunit and adjuvantapproach),” Plenum Press (NY, 1995). Pharmaceutical compositions andimmunogenic compositions within the scope of the present invention mayalso contain other compounds, which may be biologically active orinactive. For example, one or more immunogenic portions of other tumorantigens may be present, either incorporated into a fusion polypeptideor as a separate compound, within the composition.

Illustrative immunogenic compositions may contain DNA encoding one ormore of the polypeptides as described above, such that the polypeptideis generated in situ. As noted above, the DNA may be present within anyof a variety of delivery systems known to those of ordinary skill in theart, including nucleic acid expression systems, bacteria and viralexpression systems. Numerous gene delivery techniques are well known inthe art, such as those described by Rolland, Crit. Rev. Therap. DrugCarrier Systems 15:143-198, 1998, and references cited therein.Appropriate nucleic acid expression systems contain the necessary DNAsequences for expression in the patient (such as a suitable promoter andterminating signal). Bacterial delivery systems involve theadministration of a bacterium (such as Bacillus-Calmette-Guerrin) thatexpresses an immunogenic portion of the polypeptide on its cell surfaceor secretes such an epitope. In a preferred embodiment, the DNA may beintroduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus. Suitablesystems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl.Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci.569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos.4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434,1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994;Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993;Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir.Res. 73:1202-1207, 1993. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.The DNA may also be “naked,” as described, for example, in Ulmer et al.,Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells. It will be apparent that an immunogenic composition maycomprise both a polynucleotide and a polypeptide component. Suchimmunogenic compositions may provide for an enhanced immune response.

It will be apparent that an immunogenic composition may containpharmaceutically acceptable salts of the polynucleotides andpolypeptides provided herein. Such salts may be prepared frompharmaceutically acceptable non-toxic bases, including organic bases(e.g., salts of primary, secondary and tertiary amines and basic aminoacids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium,calcium and magnesium salts).

While any suitable carrier known to those of ordinary skill in the artmay be employed in the compositions of this invention, the type ofcarrier will vary depending on the mode of administration. Compositionsof the present invention may be formulated for any appropriate manner ofadministration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763;5,814,344 and 5,942,252. One may also employ a carrier comprising theparticulate-protein complexes described in U.S. Pat. No. 5,928,647,which are capable of inducing a class I-restricted cytotoxic Tlymphocyte responses in a host.

Such compositions may also comprise buffers (e.g., neutral bufferedsaline or phosphate buffered saline), carbohydrates (e.g., glucose,mannose, sucrose or dextrans), mannitol, proteins, polypeptides or aminoacids such as glycine, antioxidants, bacteriostats, chelating agentssuch as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide),solutes that render the formulation isotonic, hypotonic or weaklyhypertonic with the blood of a recipient, suspending agents, thickeningagents and/or preservatives. Alternatively, compositions of the presentinvention may be formulated as a lyophilizate. Compounds may also beencapsulated within liposomes using well known technology.

Any of a variety of immunostimulants may be employed in the immunogeniccompositions of this invention. For example, an adjuvant may beincluded. Most adjuvants contain a substance designed to protect theantigen from rapid catabolism, such as aluminum hydroxide or mineraloil, and a stimulator of immune responses, such as lipid A, Bortadellapertussis or Mycobacterium tuberculosis derived proteins. Suitableadjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2(SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminumhydroxide gel (alum) or aluminum phosphate; salts of calcium, iron orzinc; an insoluble suspension of acylated tyrosine; acylated sugars;cationically or anionically derivatized polysaccharides;polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A andquil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may alsobe used as adjuvants.

Within the immunogenic compositions provided herein, the adjuvantcomposition is preferably designed to induce an immune responsepredominantly of the Th1 type. High levels of Th1-type cytokines (e.g.,IFN-γ, TNFα, IL-2 and IL-12) tend to favor the induction of cellmediated immune responses to an administered antigen. In contrast, highlevels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend tofavor the induction of humoral immune responses. Following applicationof an immunogenic composition as provided herein, a patient will supportan immune response that includes Th1- and Th2-type responses. Within apreferred embodiment, in which a response is predominantly Th1-type, thelevel of Th1-type cytokines will increase to a greater extent than thelevel of Th2-type cytokines. The levels of these cytokines may bereadily assessed using standard assays. For a review of the families ofcytokines, see Mosmann and Coffrnan, Ann. Rev. Immunol. 7:145-173, 1989.

Preferred adjuvants for use in eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), togetherwith an aluminum salt. MPL adjuvants are available from CorixaCorporation (Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is urnethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc.,Framingham, Mass.), which may be used alone or in combination with otheradjuvants. For example, an enhanced system involves the combination of amonophosphoryl lipid A and saponin derivative, such as the combinationof QS21 and 3D-MPL as described in WO 94/00153, or a less reactogeniccomposition where the QS21 is quenched with cholesterol, as described inWO 96/33739. Other preferred formulations comprise an oil-in-wateremulsion and tocopherol. A particularly potent adjuvant formulationinvolving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion isdescribed in WO 95/17210.

Other preferred adjuvants include Montanide ISA 720 (Seppic, France),SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), theSBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available fromSmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton,Mont.), RC−529 (Corixa, Hamilton, Mont.) and other aminoalkylglucosaminide 4-phosphates (AGPs), such as those described in pendingU.S. patent application Ser. Nos. 08/853,826 and 09/074,720, thedisclosures of which are incorporated herein by reference in theirentireties.

Any immunogenic composition provided herein may be prepared using wellknown methods that result in a combination of antigen, immune responseenhancer and a suitable carrier or excipient. The compositions describedherein may be administered as part of a sustained release formulation(i.e., a formulation such as a capsule, sponge or gel (composed ofpolysaccharides, for example) that effects a slow release of compoundfollowing administration). Such formnulations may generally be preparedusing well known technology (see, e.g., Coombes et al., Vaccine14:1429-1438, 1996) and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.

Carriers for use within such formulations are biocompatible, and mayalso be biodegradable; preferably the formulation provides a relativelyconstant level of active component release. Such carriers includemicroparticles of poly(lactide-co-glycolide), polyacrylate, latex,starch, cellulose, dextran and the like. Other delayed-release carriersinclude supramolecular biovectors, which comprise a non-liquidhydrophilic core (e.g., a cross-linked polysaccharide oroligosaccharide) and, optionally, an external layer comprising anamphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO96/06638). The amount of active compound contained within a sustainedrelease formulation depends upon the site of implantation, the rate andexpected duration of release and the nature of the condition to betreated or prevented.

Any of a variety of delivery vehicles may be employed withinpharmaceutical compositions and immunogenic compositions to facilitateproduction of an antigen-specific immune response that targets tumorcells. Delivery vehicles include antigen presenting cells (APCs), suchas dendritic cells, macrophages, B cells, monocytes and other cells thatmay be engineered to be efficient APCs. Such cells may, but need not, begenetically modified to increase the capacity for presenting theantigen, to improve activation and/or maintenance of the T cellresponse, to have anti-tumor effects per se and/or to be immunologicallycompatible with the receiver (i.e., matched HLA haplotype). APCs maygenerally be isolated from any of a variety of biological fluids andorgans, including tumor and peritumoral tissues, and may be autologous,allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaive T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within an immunogenic composition (seeZitvogel et al., Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFoc to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcy receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide encoding aprostate-specific protein (or portion or other variant thereof) suchthat the prostate-specific polypeptide, or an immunogenic portionthereof, is expressed on the cell surface. Such transfection may takeplace ex vivo, and a composition comprising such transfected cells maythen be used for therapeutic purposes, as described herein.Alternatively, a gene delivery vehicle that targets a dendritic or otherantigen presenting cell may be administered to a patient, resulting intransfection that occurs in vivo. In vivo and ex vivo transfection ofdendritic cells, for example, may generally be performed using anymethods known in the art, such as those described in WO 97/24447, or thegene gun approach described by Mahvi et al., Immunology and cell Biology75:456-460, 1997. Antigen loading of dendritic cells may be achieved byincubating dendritic cells or progenitor cells with theprostate-specific polypcptide, DNA (naked or within a plasmid vector) orRNA; or with antigen-expressing recombinant bacterium or viruses (e.g.,vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading,the polypeptide may be covalently conjugated to an immunological partnerthat provides T cell help (e.g., a carrier molecule). Alternatively, adendritic cell may be pulsed with a non-conjugated immunologicalpartner, separately or in the presence of the polypeptide.

Immunogenic compositions and pharmaceutical compositions may bepresented in unit-dose or multi-dose containers, such as sealed ampoulesor vials. Such containers are preferably hermetically sealed to preservesterility of the formulation until use. In general, formulations may bestored as suspensions, solutions or emulsions in oily or aqueousvehicles. Alternatively, a immunogenic composition or pharmaceuticalcomposition may be stored in a freeze-dried condition requiring only theaddition of a sterile liquid carrier immediately prior to use.

Cancer Therapy

In further aspects of the present invention, the compositions describedherein may be used for immunotherapy of cancer, such as prostate cancer.Within such methods, pharmaceutical compositions and immunogeniccompositions are typically administered to a patient. As used herein, a“patient” refers to any warm-blooded animal, preferably a human. Apatient may or may not be afflicted with cancer. Accordingly, the abovepharmaceutical compositions and immunogenic compositions may be used toprevent the development of a cancer or to treat a patient afflicted witha cancer. A cancer may be diagnosed using criteria generally accepted inthe art, including the presence of a malignant tumor. Pharmaceuticalcompositions and immunogenic compositions may be administered eitherprior to or following surgical removal of primary tumors and/ortreatment such as administration of radiotherapy or conventionalchemotherapeutic drugs. Administration may be by any suitable method,including administration by intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal, intradermal, anal, vaginal, topical and oralroutes.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against tumors with the administration ofimmune response-modifying agents (such as polypeptides andpolynucleotides as provided herein).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedtumor-immune reactivity (such as effector cells or antibodies) that candirectly or indirectly mediate antitumor effects and does notnecessarily depend on an intact host immune system. Examples of effectorcells include T cells as discussed above, T lymphocytes (such as CD8⁺cytotoxic T lymphocytes and CD4⁺ T-helper tumor-infiltratinglymphocytes), killer cells (such as Natural Killer cells andlymphokine-activated killer cells), B cells and antigen-presenting cells(such as dendritic cells and macrophages) expressing a polypeptideprovided herein. T cell receptors and antibody receptors specific forthe polypeptides recited herein may be cloned, expressed and transferredinto other vectors or effector cells for adoptive immunotherapy. Thepolypeptides provided herein may also be used to generate antibodies oranti-idiotypic antibodies (as described above and in U.S. Pat. No.4,918,164) for passive immunotherapy.

Effector cells may generally be obtained in sufficient quantities foradoptive immunotherapy by growth in vitro, as described herein. Cultureconditions for expanding single antigen-specific effector cells toseveral billion in number with retention of antigen recognition in vivoare well known in the art. Such in vitro culture conditions typicallyuse intermittent stimulation with antigen, often in the presence ofcytokines (such as IL-2) and non-dividing feeder cells. As noted above,immunoreactive polypeptides as provided herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate asufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage, monocyte,fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., Immunological Reviews 157:177, 1997).

Alternatively, a vector expressing a polypeptide recited herein may beintroduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary, intraperitoneal or intratumor administration.

Routes and frequency of administration of the therapeutic compositionsdescribed herein, as well as dosage, will vary from individual toindividual, and may be readily established using standard techniques. Ingeneral, the pharmaceutical compositions and immunogenic compositionsmay be administered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Preferably, between 1 and 10 doses may be administered over a 52week period. Preferably, 6 doses are administered, at intervals of 1month, and booster vaccinations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients. Asuitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an anti-tumor immune response,and is at least 10-50% above the basal (i.e., untreated) level. Suchresponse can be monitored by measuring the anti-tumor antibodies in apatient or by vaccine-dependent generation of cytolytic effector cellscapable of killing the patient's tumor cells in vitro. Such immunogeniccompositions should also be capable of causing an immune response thatleads to an improved clinical outcome (e.g., more frequent remissions,complete or partial or longer disease-free survival) in treated patientsas compared to non-treated patients. In general, for pharmaceuticalcompositions and immunogenic compositions comprising one or morepolypeptides, the amount of each polypeptide present in a dose rangesfrom about 25 μg to 5 mg per kg of host. Suitable dose sizes will varywith the size of the patient, but will typically range from about 0.1 mLto about 5 mL.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., more frequent remissions, completeor partial, or longer disease-free survival) in treated patients ascompared to non-treated patients. Increases in preexisting immuneresponses to a prostate-specific protein generally correlate with animproved clinical outcome. Such immune responses may generally beevaluated using standard proliferation, cytotoxicity or cytokine assays,which may be performed using samples obtained from a patient before andafter treatment.

Cancer Detection and Diagnosis

In general, a cancer may be detected in a patient based on the presenceof one or more prostate-specific proteins and/or polynucleotidesencoding such proteins in a Ibiological sample (for example, blood,sera, sputum urine and/or tumor biopsies) obtained from the patient. Inother words, such proteins may be used as markers to indicate thepresence or absence of a cancer such as prostate cancer. In addition,such proteins may be useful for the detection of other cancers. Thebinding agents provided herein generally permit detection of the levelof antigen that binds to the agent in the biological sample.Polynucleotide primers and probes may be used to detect the level ofmRNA encoding a tumor protein, which is also indicative of the presenceor absence of a cancer. In general, a prostate-specific sequence shouldbe present at a level that is at least three fold higher in prostatetissue than in other normal tissues.

There are a variety of assay formats known to those of ordinary skill inthe art for using a binding agent to detect polypeptide markers in asample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. In general, the presence or absenceof a cancer in a patient may be determined by (a) contacting abiological sample obtained from a patient with a binding agent; (b)detecting in the sample a level of polypeptide that binds to the bindingagent; and (c) comparing the level of polypeptide with a predeterminedcut-off value.

In a preferred embodiment, the assay involves the use of binding agentimmobilized on a solid support to bind to and remove the polypeptidefrom the remainder of the sample. The bound polypeptide may then bedetected using a detection reagent that contains a reporter group andspecifically binds to the binding agent/polypeptide complex. Suchdetection reagents may comprise, for example, a binding agent thatspecifically binds to the polypeptide or an antibody or other agent thatspecifically binds to the binding agent, such as an anti-immunoglobulin,protein G, protein A or a lectin. Alternatively, a competitive assay maybe utilized, in which a polypeptide is labeled with a reporter group andallowed to bind to the immobilized binding agent after incubation of thebinding agent with the sample. The extent to which components of thesample inhibit the binding of the labeled polypeptide to the bindingagent is indicative of the reactivity of the sample with the immobilizedbinding agent. Suitable polypeptides for use within such assays includefull length prostate-specific proteins and portions thereof to which thebinding agent binds, as described above.

The solid support may be any material known to those of ordinary skillin the art to which the tumor protein may be attached. For example, thesolid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681. The binding agent may beimmobilized on the solid support using a variety of techniques known tothose of skill in the art, which are amply described in the patent andscientific literature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the agent and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In general, contacting a well of a plasticmicrotiter plate (such as polystyrene or polyvinylchloride) with anamount of binding agent ranging from about 10 ng to about 10 μg, andpreferably about 100 ng to about 1 μg, is sufficient to immobilize anadequate amount of binding agent.

Covalent attachment of binding agent to a solid support may generally beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the binding agent. For example, the bindingagent may be covalently attached to supports having an appropriatepolymer coating using benzoquinone or by condensation of an aldehydegroup on the support with an amine and an active hydrogen on the bindingpartner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991,at A 12-A13).

In certain embodiments, the assay is a two-antibody sandwich assay. Thisassay may be performed by first contacting an antibody that has beenimmobilized on a solid support, commonly the well of a microtiter plate,with the sample, such that polypeptides within the sample are allowed tobind to the immobilized antibody. Unbound sample is then removed fromthe immobilized polypeptide-antibody complexes and a detection reagent(preferably a second antibody capable of binding to a different site onthe polypeptide) containing a reporter group is added. The amount ofdetection reagent that remains bound to the solid support is thendetermined using a method appropriate for the specific reporter group.

More specifically, once the antibody is immobilized on the support asdescribed above, the remaining protein binding sites on the support aretypically blocked. Any suitable blocking agent known to those ofordinary skill in the art, such as bovine serum albumin or Tween 20™(Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is thenincubated with the sample, and polypeptide is allowed to bind to theantibody. The sample may be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is a period of timethat is sufficient to detect the presence of polypeptide within a sampleobtained from an individual with prostate cancer. Preferably, thecontact time is sufficient to achieve a level of binding that is atleast about 95% of that achieved at equilibrium between bound andunbound polypeptide. Those of ordinary skill in the art will recognizethat the time necessary to achieve equilibrium may be readily determinedby assaying the level of binding that occurs over a period of time. Atroom temperature, an incubation time of about 30 minutes is generallysufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20™. The secondantibody, which contains a reporter group, may then be added to thesolid support. Preferred reporter groups include those groups recitedabove.

The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound polypeptide. An appropriate amount of time may generally bedetermined by assaying the level of binding that occurs over a period oftime. Unbound detection reagent is then removed and bound detectionreagent is detected using the reporter group. The method employed fordetecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of a cancer, such as prostatecancer, the signal detected from the reporter group that remains boundto the solid support is generally compared to a signal that correspondsto a predetermined cut-off value. In one preferred embodiment, thecut-off value for the detection of a cancer is the average mean signalobtained when the immobilized antibody is incubated with samples frompatients without the cancer. In general, a sample generating a signalthat is three standard deviations above the predetermined cut-off valueis considered positive for the cancer. In an alternate preferredembodiment, the cut-off value is determined using a Receiver OperatorCurve, according to the method of Sackett et al., Clinical Epidemiology:A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p.106-7. Briefly, in this embodiment, the cut-off value may be determinedfrom a plot of pairs of true positive rates (i.e., sensitivity) andfalse positive rates (100%-specificity) that correspond to each possiblecut-off value for the diagnostic test result. The cut-off value on theplot that is the closest to the upper left-hand corner (i.e., the valuethat encloses the largest area) is the most accurate cut-off value, anda sample generating a signal that is higher than the cut-off valuedetermined by this method may be considered positive. Alternatively, thecut-off value may be shifted to the left along the plot, to minimize thefalse positive rate, or to the right, to minimize the false negativerate. In general, a sample generating a signal that is higher than thecut-off value determined by this method is considered positive for acancer.

In a related embodiment, the assay is performed in a flow-through orstrip test format, wherein the binding agent is immobilized on amembrane, such as nitrocellulose. In the flow-through test, polypeptideswithin the sample bind to the immobilized binding agent as the samplepasses through the membrane. A second, labeled binding agent then bindsto the binding agent-polypeptide complex as a solution containing thesecond binding agent flows through the membrane. The detection of boundsecond binding agent may then be performed as described above. In thestrip test format, one end of the membrane to which binding agent isbound is immersed in a solution containing the sample. The samplemigrates along the membrane through a region containing second bindingagent and to the area of immobilized binding agent. Concentration ofsecond binding agent at the area of immobilized antibody indicates thepresence of a cancer. Typically, the concentration of second bindingagent at that site generates a pattern, such as a line, that can be readvisually. The absence of such a pattern indicates a negative result. Ingeneral, the amount of binding agent immobilized on the membrane isselected to generate a visually discernible pattern when the biologicalsample contains a level of polypeptide that would be sufficient togenerate a positive signal in the two-antibody sandwich assay, in theformat discussed above. Preferred binding agents for use in such assaysare antibodies and antigen-binding fragments thereof. Preferably, theamount of antibody immobilized on the membrane ranges from about 25 ngto about 1 μg, and more preferably from about 50 ng to about 500 ng.Such tests can typically be performed with a very small amount ofbiological sample.

Of course, numerous other assay protocols exist that are suitable foruse with the tumor proteins or binding agents of the present invention.The above descriptions are intended to be exemplary only. For example,it will be apparent to those of ordinary skill in the art that the aboveprotocols may be readily modified to use prostate-specific polypeptidesto detect antibodies that bind to such polypeptides in a biologicalsample. The detection of such prostate-specific protein specificantibodies may correlate with the presence of a cancer.

A cancer may also, or alternatively, be detected based on the presenceof T cells that specifically react with a prostate-specific protein in abiological sample. Within certain methods, a biological samplecomprising CD4⁺ and/or CD8⁺ T cells isolated from a patient is incubatedwith a prostate-specific polypeptide, a polynucleotide encoding such apolypeptide and/or an APC that expresses at least an immunogenic portionof such a polypeptide, and the presence or absence of specificactivation of the T cells is detected. Suitable biological samplesinclude, but are not limited to, isolated T cells. For example, T cellsmay be isolated from a patient by routine techniques (such as byFicoll/Hypaque density gradient centrifugation of peripheral bloodlymphocytes). T cells may be incubated in vitro for 2-9 days (typically4 days) at 37° C. with polypeptide (e.g., 5-25 μg/ml). It be desirableto incubate another aliquot of a T cell sample in the absence ofprostate-specific polypeptide to serve as a control. For CD4⁺ T cells,activation is preferably detected by evaluating proliferation of the Tcells. For CD8⁺ T cells, activation is preferably detected by evaluatingcytolytic activity. A level of proliferation that is at least two foldgreater and/or a level of cytolytic activity that is at least 20%greater than in disease-free patients indicates the presence of a cancerin the patient.

As noted above, a cancer may also, or alternatively, be detected basedon the level of mRNA encoding a prostate-specific protein in abiological sample. For example, at least two oligonucleotide primers maybe employed in a polymerase chain reaction (PCR) based assay to amplifya portion of a prostate-specific cDNA derived from a biological sample,wherein at least one of the oligonucleotide primers is specific for(i.e., hybridizes to) a polynucleotide encoding the prostate-specificprotein. The amplified cDNA is then separated and detected usingtechniques well known in the art, such as gel electrophoresis.Similarly, oligonucleotide probes that specifically hybridize to apolynucleotide encoding a prostate-specific protein may be used in ahybridization assay to detect the presence of polynucleotide encodingthe tumor protein in a biological sample.

To permit hybridization under assay conditions, oligonucleotide primersand probes should comprise an oligonucleotide sequence that has at leastabout 60%, preferably at least about 75% and more preferably at leastabout 90%, identity to a portion of a polynucleotide encoding aprostate-specific protein that is at least 10 nucleotides, andpreferably at least 20 nucleotides, in length. Preferably,oligonucleotide primers and/or probes hybridize to a polynucleotideencoding a polypeptide described herein under moderately stringentconditions, as defined above. Oligonucleotide primers and/or probeswhich may be usefully employed in the diagnostic methods describedherein preferably are at least 10-40 nucleotides in length. In apreferred embodiment, the oligonucleotide primers comprise at least 10contiguous nucleotides, more preferably at least 15 contiguousnucleotides, of a DNA molecule having a sequence recited in SEQ ID NO:1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335,340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552,569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823 and824. Techniques for both PCR based assays and hybridization assays arewell known in the art (see, for example, Mullis et al., Cold SpringHarbor Symp. Quant. Biol., 51:263, 1987; Erlich ed., PCR Technology,Stockton Press, N.Y., 1989).

One preferred assay employs RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma biological sample, such as biopsy tissue, and is reverse transcribedto produce cDNA molecules. PCR amplification using at least one specificprimer generates a cDNA molecule, which may be separated and visualizedusing, for example, gel electrophoresis. Amplification may be performedon biological samples taken from a test patient and from an individualwho is not afflicted with a cancer. The amplification reaction may beperformed on several dilutions of cDNA spanning two orders of magnitude.A two-fold or greater increase in expression in several dilutions of thetest patient sample as compared to the same dilutions of thenon-cancerous sample is typically considered positive.

In another embodiment, the compositions described herein may be used asmarkers for the progression of cancer. In this embodiment, assays asdescribed above for the diagnosis of a cancer may be performed overtime, and the change in the level of reactive polypeptide(s) orpolynucleotide(s) evaluated. For example, the assays may be performedevery 24-72 hours for a period of 6 months to 1 year, and thereafterperformed as needed. In general, a cancer is progressing in thosepatients in whom the level of polypeptide or polynucleotide detectedincreases over time. In contrast, the cancer is not progressing when thelevel of reactive polypeptide or polynucleotide either remains constantor decreases with time.

Certain in vivo diagnostic assays may be performed directly on a tumor.One such assay involves contacting tumor cells with a binding agent. Thebound binding agent may then be detected directly or indirectly via areporter group. Such binding agents may also be used in histologicalapplications. Alternatively, polynucleotide probes may be used withinsuch applications.

As noted above, to improve sensitivity, multiple prostate-specificprotein markers may be assayed within a given sample. It will beapparent that binding agents specific for different proteins providedherein may be combined within a single assay. Further, multiple primersor probes may be used concurrently. The selection of tumor proteinmarkers may be based on routine experiments to determine combinationsthat results in optimal sensitivity. In addition, or alternatively,assays for tumor proteins provided herein may be combined with assaysfor other known tumor antigens.

Diagnostic Kits

The present invention further provides kits for use within any of theabove diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to a prostate-specific protein. Suchantibodies or fragments may be provided attached to a support material,as described above. One or more additional containers may encloseelements, such as reagents or buffers, to be used in the assay. Suchkits may also, or alternatively, contain a detection reagent asdescribed above that contains a reporter group suitable for direct orindirect detection of antibody binding.

Alternatively, a kit may be designed to detect the level of mRNAencoding a prostate-specific protein in a biological sample. Such kitsgenerally comprise at least one oligonucleotide probe or primer, asdescribed above, that hybridizes to a polynucleotide encoding aprostate-specific protein. Such an oligonucleotide may be used, forexample, within a PCR or hybridization assay. Additional components thatmay be present within such kits include a second oligonucleotide and/ora diagnostic reagent or container to facilitate the detection of apolynucleotide encoding a prostate-specific protein.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 Isolation and Characterization of Prostate-sperificPolypeptides

This Example describes the isolation of certain prostate-specificpolypeptides from a prostate tumor cDNA library.

A human prostate tumor cDNA expression library was constructed fromprostate tumor poly A⁺ RNA using a Superscript Plasmid System for cDNASynthesis and Plasmid Cloning kit (BRL Life Technologies, Gaithersburg,Md. 20897) following the manufacturer's protocol. Specifically, prostatetumor tissues were homogenized with polytron (Kinematica, Switzerland)and total RNA was extracted using Trizol reagent (BRL Life Technologies)as directed by the manufacturer. The poly A⁺ RNA was then purified usinga Qiagen oligotex spin column mRNA purification kit (Qiagen, SantaClarita, Calif. 91355) according to the manufacturer's protocol.First-strand cDNA was synthesized using the NotI/Oligo-dT18 primer.Double-stranded cDNA was synthesized, ligated with EcoRI/BAXI adaptors(Invitrogen, San Diego, Calif.) and digested with NotI. Following sizefractionation with Chroma Spin-1000 columns (Clontech, Palo Alto,Calif.), the cDNA was ligated into the EcoRI/NotI site of pCDNA3.1(Invitrogen) and transformed into ElectroMax E. coli DH10B cells (BRLLife Technologies) by electroporation.

Using the same procedure, a normal human pancreas cDNA expressionlibrary was prepared from a pool of six tissue specimens (Clontech). ThecDNA libraries were characterized by determining the number ofindependent colonies, the percentage of clones that carried insert, theaverage insert size and by sequence analysis. The prostate tumor librarycontained 1.64×10⁷ independent colonies, with 70% of clones having aninsert and the average insert size being 1745 base pairs. The normalpancreas cDNA library contained 3.3×10⁶ independent colonies, with 69%of clones having inserts and the average insert size being 1120 basepairs. For both libraries, sequence analysis showed that the majority ofclones had a full length cDNA sequence and were synthesized from mRNA,with minimal rRNA and mitochondrial DNA contamination.

cDNA library subtraction was performed using the above prostate tumorand normal pancreas cDNA libraries, as described by Hara et al. (Blood,84:189-199, 1994) with some modifications. Specifically, a prostatetumor-specific subtracted cDNA library was generated as follows. Normalpancreas cDNA library (70 μg) was digested with EcoRI, NotI, and SfuI,followed by a filling-in reaction with DNA polymerase Klenow fragment.After phenol-chloroform extraction and ethanol precipitation, the DNAwas dissolved in 100 μl of H₂O, heat-denatured and mixed with 100 μl(100 μg) of Photoprobe biotin (Vector Laboratories, Burlingame, Calif.).As recommended by the manufacturer, the resulting mixture was irradiatedwith a 270 W sunlamp on ice for 20 minutes. Additional Photoprobe biotin(50 μl ) was added and the biotinylation reaction was repeated. Afterextraction with butanol five times, the DNA was ethanol-precipitated anddissolved in 23 μl H₂O to form the driver DNA.

To form the tracer DNA, 10 μg prostate tumor cDNA library was digestedwith BamHI and XhoI, phenol chloroform extracted and passed throughChroma spin-400 columns (Clontech). Following ethanol precipitation, thetracer DNA was dissolved in 5 μl H₂O. Tracer DNA was mixed with 15 μldriver DNA and 20 μl of 2×hybridization buffer (1.5 M NaCl/10 mM EDTA/50mM HEPES pH 7.5/0.2% sodium dodecyl sulfate), overlaid with mineral oil,and heat-denatured completely. The sample was immediately transferredinto a 68° C. water bath and incubated for 20 hours (long hybridization[LH]). The reaction mixture was then subjected to a streptavidintreatment followed by phenol/chloroform extraction. This process wasrepeated three more times. Subtracted DNA was precipitated, dissolved in12 μl H₂O, mixed with 8 μl driver DNA and 20 μl of 2×hybridizationbuffer, and subjected to a hybridization at 68° C. for 2 hours (shorthybridization [SH]). After removal of biotinylated double-stranded DNA,subtracted cDNA was ligated into BamHI/XhoI site of chloramphenicolresistant pBCSK⁺ (Stratagene, La Jolla, Calif. 92037) and transformedinto ElectroMax E. coli DH10B cells by electroporation to generate aprostate tumor specific subtracted cDNA library (referred to as“prostate subtraction 1”).

To analyze the subtracted cDNA library, plasmid DNA was prepared from100 independent clones, randomly picked from the subtracted prostatetumor specific library and grouped based on insert size. RepresentativecDNA clones were further characterized by DNA sequencing with a PerkinElmer/Applied Biosystems Division Automated Sequencer Model 373A (FosterCity, Calif.). Six cDNA clones, hereinafter referred to as F1-13, F1-12,F1-16, H1-1, H1-9 and H1-4, were shown to be abundant in the subtractedprostate-specific cDNA library. The determined 3′ and 5′ cDNA sequencesfor F1-12 are provided in SEQ ID NO: 2 and 3, respectively, withdetermined 3′ cDNA sequences for F1-13, F1-16, H1-1, H1-9 and H1-4 beingprovided in SEQ ID NO: 1 and 4-7, respectively.

The cDNA sequences for the isolated clones were compared to knownsequences in the gene bank using the EMBL and GenBank databases (release96). Four of the prostate tumor cDNA clones, F1-13, F1-16, H1-1, andH1-4, were determined to encode the following previously identifiedproteins: prostate specific antigen (PSA), human glandular kallikrein,human tumor expression enhanced gene, and mitochondria cytochrome Coxidase subunit II. H1-9 was found to be identical to a previouslyidentified human autonomously replicating sequence. No significanthomologies to the cDNA sequence for F1-12 were found.

Subsequent studies led to the isolation of a full-length cDNA sequencefor F1-12 (also referred to as P504S). This sequence is provided in SEQID NO: 107, with the corresponding predicted amino acid sequence beingprovided in SEQ ID NO: 108. cDNA splice variants of P504S are providedin SEQ ID NO: 600-605.

To clone less abundant prostate tumor specific genes, cDNA librarysubtraction was performed by subtracting the prostate tumor cDNA librarydescribed above with the normal pancreas cDNA library and with the threemost abundant genes in the previously subtracted prostate tumor specificcDNA library: human glandular kallikrein, prostate specific antigen(PSA), and mitochondria cytochrome C oxidase subunit II. Specifically, 1μg each of human glandular kallikrein, PSA and mitochondria cytochrome Coxidase subunit II cDNAs in pCDNA3.1 were added to the driver DNA andsubtraction was performed as described above to provide a secondsubtracted cDNA library hereinafter referred to as the “subtractedprostate tumor specific cDNA library with spike”.

Twenty-two cDNA clones were isolated from the subtracted prostate tumorspecific cDNA library with spike. The determined 3′ and 5′ cDNAsequences for the clones referred to as J1-17, L1-12, N1-1862, J1-13,J1-19, J1-25, J1-24, K1-58, K1-63, L1-4 and L1-14 are provided in SEQ IDNOS: 8-9, 10-11, 12-13, 14-15, 16-17, 18-19, 20-21, 22-23, 24-25, 26-27and 28-29, respectively. The determined 3′ cDNA sequences for the clonesreferred to as J1-12, J1-16, J1-21, K1-48, K1-55, L1-2, L1-6, N1-1858,N1-1860, N1-1861, N1-1864 are provided in SEQ ID NOS: 30-40,respectively. Comparison of these sequences with those in the gene bankas described above, revealed no significant homologies to three of thefive most abundant DNA species, (J1-17, L1-12 and N1-1862; SEQ ID NOS:8-9, 10-11 and 12-13, respectively). Of the remaining two most abundantspecies, one (J1-12; SEQ ID NO:30) was found to be identical to thepreviously identified human pulmonary surfactant-associated protein, andthe other (K1-48; SEQ ID NO:33) was determined to have some homology toR. norvegicus mRNA for 2-arylpropionyl-CoA epimerase. Of the 17 lessabundant cDNA clones isolated from the subtracted prostate tumorspecific cDNA library with spike, four (J1-16, K1-55, L1-6 and N1-1864;SEQ ID NOS:31, 34, 36 and 40, respectively) were found to be identicalto previously identified sequences, two (J1-21 and N1-1860; SEQ ID NOS:32 and 38, respectively) were found to show some homology to non-humansequences, and two (L1-2 and N1-1861; SEQ ID NOS: 35 and 39,respectively) were found to show some homology to known human sequences.No significant homologies were found to the polypeptides J1-13, J1-19,J1-24, J1-25, K1-58, K1-63, L1-4, L1-14 (SEQ ID NOS: 14-15, 16-17,20-21, 18-19, 22-23, 24-25, 26-27, 28-29, respectively).

Subsequent studies led to the isolation of full length cDNA sequencesfor J1-17, L1-12 and N1-1862 (SEQ ID NOS: 109-111, respectively). Thecorresponding predicted amino acid sequences are provided in SEQ ID NOS:112-114. L1-12 is also referred to as P501S. A cDNA splice variant ofP501S is provided in SEQ ID NO: 606.

In a further experiment, four additional clones were identified bysubtracting a prostate tumor cDNA library with normal prostate cDNAprepared from a pool of three normal prostate poly A+ RNA (referred toas “prostate subtraction 2”). The determined cDNA sequences for theseclones, hereinafter referred to as U1-3064, U1-3065, V1-3692 and1A-3905, are provided in SEQ ID NO: 69-72, respectively. Comparison ofthe determined sequences with those in the gene bank revealed nosignificant homologies to U1-3065.

A second subtraction with spike (referred to as “prostate subtractionspike 2”) was performed by subtracting a prostate tumor specific cDNAlibrary with spike with normal pancreas cDNA library and further spikedwith PSA, J1-17, pulmonary surfactant-associated protein, mitochondrialDNA, cytochrome c oxidase subunit II, N1-1862, autonomously replicatingsequence, L1-12 and tumor expression enhanced gene. Four additionalclones, hereinafter referred to as V1-3686, R1-2330, 1B-3976 andV1-3679, were isolated. The determined cDNA sequences for these clonesare provided in SEQ ID NO:73-76, respectively. Comparison of thesesequences with those in the gene bank revealed no significant homologiesto V1-3686 and R1-2330.

Further analysis of the three prostate subtractions described above(prostate subtraction 2, subtracted prostate tumor specific cDNA librarywith spike, and prostate subtraction spike 2) resulted in theidentification of sixteen additional clones, referred to as 1G-4736,1G-4738, 1G-4741, 1G-4744, 1G-4734, 1H-4774, 1H-4781, 1H-4785, 1H-4787,1H-4796, 1I-4810, 1I-4811, 1J-4876, 1K-4884 and 1K-4896. The determinedcDNA sequences for these clones are provided in SEQ ID NOS: 77-92,respectively. Comparison of these sequences with those in the gene bankas described above, revealed no significant homologies to 1G-4741,1G-4734, 1I-4807, 1J-4876 and 1K-4896 (SEQ ID NOS: 79, 81, 87, 90 and92, respectively). Further analysis of the isolated clones led to thedetermination of extended cDNA sequences for 1G-4736, 1G-4738, 1G-4741,1G-4744, 1H-4774, 1H-4781, 1H-4785, 1H-4787, 1H-4796, 1I-4807, 1J-4876,1K-4884 and 1K-4896, provided in SEQ ID NOS: 179-188 and 191-193,respectively, and to the determination of additional partial cDNAsequences for 1I-4810 and 1I-4811, provided in SEQ ID NOS: 189 and 190,respectively.

Additional studies with prostate subtraction spike 2 resulted in theisolation of three more clones. Their sequences were determined asdescribed above and compared to the most recent GenBank. All threeclones were found to have homology to known genes, which areCysteine-rich protein, KIAA0242, and KIAA0280 (SEQ ID NO: 317, 319, and320, respectively). Further analysis of these clones by Syntenimicroarray (Synteni, Palo Alto, Calif.) demonstrated that all threeclones were over-expressed in most prostate tumors and prostate BPH, aswell as in the majority of normal prostate tissues tested, but lowexpression in all other normal tissues.

An additional subtraction was performed by subtracting a normal prostatecDNA library with normal pancreas cDNA (referred to as “prostatesubtraction 3”). This led to the identification of six additional clonesreferred to as 1G-4761, 1G-4762, 1H-4766, 1H-4770, 1H-4771 and 1H-4772(SEQ ID NOS: 93-98). Comparison of these sequences with those in thegene bank revealed no significant homologies to 1G-4761 and 1H-4771 (SEQID NOS: 93 and 97, respectively). Further analysis of the isolatedclones led to the determination of extended cDNA sequences for 1G-4761,1G-4762, 1H-4766 and 1H-4772 provided in SEQ ID NOS: 194-196 and 199,respectively, and to the determination of additional partial cDNAsequences for 1H-4770 and 1H-4771, provided in SEQ ID NOS: 197 and 198,respectively.

Subtraction of a prostate tumor cDNA library, prepared from a pool ofpolyA+ RNA from three prostate cancer patients, with a normal pancreascDNA library (prostate subtraction 4) led to the identification of eightclones, referred to as 1D-4297, 1D-4309, 1D.1-4278, 1D-4288, 1D-4283,1D-4304, 1D-4296 and 1D-4280 (SEQ ID NOS: 99-107). These sequences werecompared to those in the gene bank as described above. No significanthomologies were found to 1D-4283 and 1D-4304 (SEQ ID NOS: 103 and 104,respectively). Further analysis of the isolated clones led to thedetermination of extended cDNA sequences for 1D-4309, 1D.1-4278,1D-4288, 1D-4283, 1D-4304, 1D-4296 and 1D-4280, provided in SEQ ID NOS:200-206, respectively. cDNA clones isolated in prostate subtraction 1and prostate subtraction 2, described above, were colony PCR amplifiedand their mRNA expression levels in prostate tumor, normal prostate andin various other normal tissues were determined using microarraytechnology (Synteni, Palo Alto, Calif.). Briefly, the PCR amplificationproducts were dotted onto slides in an array format, with each productoccupying a unique location in the array. mRNA was extracted from thetissue sample to be tested, reverse transcribed, and fluorescent-labeledcDNA probes were generated. The microarrays were probed with the labeledcDNA probes, the slides scanned and fluorescence intensity was measured.This intensity correlates with the hybridization intensity. Two clones(referred to as P509S and P510S) were found to be over-expressed inprostate tumor and normal prostate and expressed at low levels in allother normal tissues tested (liver, pancreas, skin, bone marrow, brain,breast, adrenal gland, bladder, testes, salivary gland, large intestine,kidney, ovary, lung, spinal cord, skeletal muscle and colon). Thedetermined cDNA sequences for P509S and p510S are provided in SEQ ID NO:223 and 224, respectively. Comparison of these sequences with those inthe gene bank as described above, revealed some homology to previouslyidentified ESTs.

Additional, studies led to the isolation of the full-length cDNAsequence for P509S. This sequence is provided in SEQ ID NO: 332, withthe corresponding predicted amino acid sequence being provided in SEQ IDNO: 339. Two variant full-length cDNA sequences for P510S are providedin SEQ ID NO: 535 and 536, with the corresponding predicted amino acidsequences being provided in SEQ ID NO: 537 and 538, respectively.Additional splice variants of p510S are provided in SEQ ID NO: 598 and599.

The determined cDNA sequences for additional prostate-specific clonesisolated during characterization of prostate specific cDNA libraries areprovided in SEQ ID NO: 618-689, 691-697 and 709-772. Comparison of thesesequences with those in the public databases revealed no significanthomologies to any of these sequences.

EXAMPLE 2 Determination of Tissue Specificity of Prostate-specificPolypeptides

Using gene specific primers, mRNA expression levels for therepresentative prostate-specific polypeptides F1-16, H1-1, J1-17 (alsoreferred to as P502S), L1-12 (also referred to as P501S), F1-12 (alsoreferred to as P504S) and N1-1862 (also referred to as P503S) wereexamined in a variety of normal and tumor tissues using RT-PCR.

Briefly, total RNA was extracted from a variety of normal and tumortissues using Trizol reagent as described above. First strand synthesiswas carried out using 1-2 μg of total RNA with SuperScript II reversetranscriptase (BRL Life Technologies) at 42° C. for one hour. The cDNAwas then amplified by PCR with gene-specific primers. To ensure thesemi-quantitative nature of the RT-PCR, β-actin was used as an internalcontrol for each of the tissues examined. First, serial dilutions of thefirst strand cDNAs were prepared and RT-PCR assays were performed usingβ-actin specific primers. A dilution was then chosen that enabled thelinear range amplification of the β-actin template and which wassensitive enough to reflect the differences in the initial copy numbers.Using these conditions, the β-actin levels were determined for eachreverse transcription reaction from each tissue. DNA contamination wasminimized by DNase treatment and by assuring a negative PCR result whenusing first strand cDNA that was prepared without adding reversetranscriptase.

mRNA Expression levels were examined in four different types of tumortissue (prostate tumor from 2 patients, breast tumor from 3 patients,colon tumor, lung tumor), and sixteen different normal tissues,including prostate, colon, kidney, liver, lung, ovary, pancreas,skeletal muscle, skin, stomach, testes, bone marrow and brain. F1-16 wasfound to be expressed at high levels in prostate tumor tissue, colontumor and normal prostate, and at lower levels in normal liver, skin andtestes, with expression being undetectable in the other tissuesexamined. H1-1 was found to be expressed at high levels in prostatetumor, lung tumor, breast tumor, normal prostate, normal colon andnormal brain, at much lower levels in normal lung, pancreas, skeletalmuscle, skin, small intestine, bone marrow, and was not detected in theother tissues tested. J1-17 (P502S) and L1-12 (P501S) appear to bespecifically over-expressed in prostate, with both genes being expressedat high levels in prostate tumor and normal prostate but at low toundetectable levels in all the other tissues examined. N1-1862 (P503S)was found to be over-expressed in 60% of prostate tumors and detectablein normal colon and kidney. The RT-PCR results thus indicate that F1-16,H1-1, J1-17 (P502S), N1-1862 (P503S) and L1-12 (P501S) are eitherprostate specific or are expressed at significantly elevated levels inprostate.

Further RT-PCR studies showed that F1-12 (P504S) is over-expressed in60% of prostate tumors, detectable in normal kidney but not detectablein all other tissues tested. Similarly, R1-2330 was shown to beover-expressed in 40% of prostate tumors, detectable in normal kidneyand liver, but not detectable in all other tissues tested. U1-3064 wasfound to be over-expressed in 60% of prostate tumors, and also expressedin breast and colon tumors, but was not detectable in normal tissues.

RT-PCR characterization of R1-2330, U1-3064 and 1D-4279 showed thatthese three antigens are over-expressed in prostate and/or prostatetumors.

Northern analysis with four prostate tumors, two normal prostatesamples, two BPH prostates, and normal colon, kidney, liver, lung,pancrease, skeletal muscle, brain, stomach, testes, small intestine andbone marrow, showed that L1-12 (P501S) is over-expressed in prostatetumors and normal prostate, while being undetectable in other normaltissues tested. J1-17 (P502S) was detected in two prostate tumors andnot in the other tissues tested. N1-1862 (P503S) was found to beover-expressed in three prostate tumors and to be expressed in normalprostate, colon and kidney, but not in other tissues tested. F1-12(P504S) was found to be highly expressed in two prostate tumors and tobe undetectable in all other tissues tested.

The microarray technology described above was used to determine theexpression levels of representative antigens described herein inprostate tumor, breast tumor and the following normal tissues: prostate,liver, pancreas, skin, bone marrow, brain, breast, adrenal gland,bladder, testes, salivary gland, large intestine, kidney, ovary, lung,spinal cord, skeletal muscle and colon. L1-12 (P501S) was found to beover-expressed in normal prostate and prostate tumor, with someexpression being detected in normal skeletal muscle. Both J1-12 andF1-12 (P504S) were found to be over-expressed in prostate tumor, withexpression being lower or undetectable in all other tissues tested.N1-1862 (P503S) was found to be expressed at high levels in prostatetumor and normal prostate, and at low levels in normal large intestineand normal colon, with expression being undetectable in all othertissues tested. R1-2330 was found to be over-expressed in prostate tumorand normal prostate, and to be expressed at lower levels in all othertissues tested. 1D-4279 was found to be over-expressed in prostate tumorand normal prostate, expressed at lower levels in normal spinal cord,and to be undetectable in all other tissues tested.

Further microarray analysis to specifically address the extent to whichP501S (SEQ ID NO: 110) was expressed in breast tumor revealed moderateover-expression not only in breast tumor, but also in metastatic breasttumor (2/31), with negligible to low expression in normal tissues. Thisdata suggests that P501S may be over-expressed in various breast tumorsas well as in prostate tumors.

The expression levels of 32 ESTs (expressed sequence tags) described byVasmatzis et al. (Proc. Natl. Acad. Sci. USA 95:300-304, 1998) in avariety of tumor and normal tissues were examined by microarraytechnology as described above. Two of these clones (referred to asP1000C and P1001C) were found to be over-expressed in prostate tumor andnormal prostate, and expressed at low to undetectable levels in allother tissues tested (normal aorta, thymus, resting and activated PBMC,epithelial cells, spinal cord, adrenal gland, fetal tissues, skin,salivary gland, large intestine, bone marrow, liver, lung, dendriticcells, stomach, lymph nodes, brain, heart, small intestine, skeletalmuscle, colon and kidney. The determined cDNA sequences for P1000C andP1001C are provided in SEQ ID NO: 384 and 472, respectively. Thesequence of P1001C was found to show some homology to the previouslyisolated Human mRNA for JM27 protein. No significant homologies werefound to the sequence of P1000C.

The expression of the polypeptide encoded by the full length cDNAsequence for F1-12 (also referred to as P504S; SEQ ID NO: 108) wasinvestigated by immunohistochemical analysis. Rabbit-anti-P504Spolyclonal antibodies were generated against the full length P504Sprotein by standard techniques. Subsequent isolation andcharacterization of the polyclonal antibodies were also performed bytechniques well known in the art. Immunohistochemical analysis showedthat the P504S polypeptide was expressed in 100% of prostate carcinomasamples tested (n=5).

The rabbit-anti-P504S polyclonal antibody did not appear to label benignprostate cells with the same cytoplasmic granular staining, but ratherwith light nuclear staining. Analysis of normal tissues revealed thatthe encoded polypeptide was found to be expressed in some, but not allnormal human tissues. Positive cytoplasmic staining withrabbit-anti-P504S polyclonal antibody was found in normal human kidney,liver, brain, colon and lung-associated macrophages, whereas heart andbone marrow were negative.

This data indicates that the P504S polypeptide is present in prostatecancer tissues, and that there are qualitative and quantitativedifferences in the staining between benign prostatic hyperplasia tissuesand prostate cancer tissues, suggesting that this polypeptide may bedetected selectively in prostate tumors and therefore be usefuil in thediagnosis of prostate cancer.

EXAMPLE 3 Isolation and Characterization of Prostate-specificPolypeptides by Pcr-based Subtraction

A cDNA subtraction library, containing cDNA from normal prostatesubtracted with ten other normal tissue cDNAs (brain, heart, kidney,liver, lung, ovary, placenta, skeletal muscle, spleen and thymus) andthen submitted to a first round of PCR amplification, was purchased fromClontech. This library was subjected to a second round of PCRamplification, following the manufacturer's protocol. The resulting cDNAfragments were subcloned into the vector pT7 Blue T-vector (Novagen,Madison, Wis.) and transformed into XL-1 Blue MRF′ E. coli (Stratagene).DNA was isolated from independent clones and sequenced using a PerkinElmer/Applied Biosystems Division Automated Sequencer Model 373A.

Fifty-nine positive clones were sequenced. Comparison of the DNAsequences of these clones with those in the gene bank, as describedabove, revealed no significant homologies to 25 of these clones,hereinafter referred to as P5, P8, P9, P18, P20, P30, P34, P36, P38,P39, P42, P49, P50, P53, P55, P60, P64, P65, P73, P75, P76, P79 and P84.The determined cDNA sequences for these clones are provided in SEQ IDNO: 41-45, 47-52 and 54-65, respectively. P29, P47, P68, P80 and P82(SEQ ID NO: 46, 53 and 66-68, respectively) were found to show somedegree of homology to previously identified DNA sequences. To the bestof the inventors' knowledge, none of these sequences have beenpreviously shown to be present in prostate.

Further studies employing the sequence of SEQ ID NO: 67 as a probe instandard full-length cloning methods, resulted in the isolation of threecDNA sequences which appear to be splice variants of P80 (also known asP704P). These sequences are provided in SEQ ID NO: 699-701.

Further studies using the PCR-based methodology described above resultedin the isolation of more than 180 additional clones, of which 23 cloneswere found to show no significant homologies to known sequences. Thedetermined cDNA sequences for these clones are provided in SEQ ID NO:115-123, 127, 131, 137, 145, 147-151, 153, 156-158 and 160. Twenty-threeclones (SEQ ID NO: 124-126, 128-130, 132-136, 138-144, 146, 152, 154,155 and 159) were found to show some homology to previously identifiedESTs. An additional ten clones (SEQ ID NO: 161-170) were found to havesome degree of homology to known genes. Larger cDNA clones containingthe P20 sequence represent splice variants of a gene referred to asP703P. The determined DNA sequence for the variants referred to as DE1,DE13 and DE14 are provided in SEQ ID NOS: 171, 175 and 177,respectively, with the corresponding predicted amino acid sequencesbeing provided in SEQ ID NO: 172, 176 and 178, respectively. Thedetermined cDNA sequence for an extended spliced form of P703 isprovided in SEQ ID NO: 225. The DNA sequences for the splice variantsreferred to as DE2 and DE6 are provided in SEQ ID NOS: 173 and 174,respectively.

mRNA Expression levels for representative clones in tumor tissues(prostate (n=5), breast (n=2), colon and lung) normal tissues (prostate(n=5), colon, kidney, liver, lung (n=2), ovary (n=2), skeletal muscle,skin, stomach, small intestine and brain), and activated andnon-activated PBMC was determined by RT-PCR as described above.Expression was examined in one sample of each tissue type unlessotherwise indicated.

P9 was found to be highly expressed in normal prostate and prostatetumor compared to all normal tissues tested except for normal colonwhich showed comparable expression. P20, a portion of the P703P gene,was found to be highly expressed in normal prostate and prostate tumor,compared to all twelve normal tissues tested. A modest increase inexpression of P20 in breast tumor (n=2), colon tumor and lung tumor wasseen compared to all normal tissues except lung (1 of 2). Increasedexpression of P18 was found in normal prostate, prostate tumor andbreast tumor compared to other normal tissues except lung and stomach. Amodest increase in expression of P5 was observed in normal prostatecompared to most other normal tissues. However, some elevated expressionwas seen in normal lung and PBMC. Elevated expression of P5 was alsoobserved in prostate tumors (2 of 5), breast tumor and one lung tumorsample. For P30, similar expression levels were seen in normal prostateand prostate tumor, compared to six of twelve other normal tissuestested. Increased expression was seen in breast tumors, one lung tumorsample and one colon tumor sample, and also in normal PBMC. P29 wasfound to be over-expressed in prostate tumor (5 of 5) and normalprostate (5 of 5) compared to the majority of normal tissues. However,substantial expression of P29 was observed in normal colon and normallung (2 of 2). P80 was found to be over-expressed in prostate tumor (5of 5) and normal prostate (5 of 5) compared to all other normal tissuestested, with increased expression also being seen in colon tumor.

Further studies resulted in the isolation of twelve additional clones,hereinafter referred to as 10-d8, 10-h10, 11-c8, 7-g6, 8-b5, 8-b6, 8-d4,8-d9, 8-g3, 8-h11, 9-f12 and 9-f3. The determined DNA sequences for10-d8, 10-h10, 11-c8, 8-d4, 8-d9, 8-h11, 9-f12 and 9-f3 are provided inSEQ ID NO: 207, 208, 209, 216, 217, 220, 221 and 222, respectively. Thedetermined forward and reverse DNA sequences for 7-g6, 8-b5, 8-b6 and8-g3 are provided in SEQ ID NO: 210 and 211; 212 and 213; 214 and 215;and 218 and 219, respectively. Comparison of these sequences with thosein the gene bank revealed no significant homologies to the sequence of9-f3. The clones 10-d8, 11-c8 and 8-h11 were found to show some homologyto previously isolated ESTs, while 10-h10, 8-b5, 8-b6, 8-d4, 8-d9, 8-g3and 9-f12 were found to show some homology to previously identifiedgenes. Further characterization of 7-G6 and 8-G3 showed identity to theknown genes PAP and PSA, respectively.

mRNA expression levels for these clones were determined using themicro-array technology described above. The clones 7-G6, 8-G3, 8-B5,8-B6, 8-D4, 8-D9, 9-F3, 9-F12, 9-H3, 10-A2, 10-A4, 11-C9 and 11-F2 werefound to be over-expressed in prostate tumor and normal prostate, withexpression in other tissues tested being low or undetectable. Increasedexpression of 8-F11 was seen in prostate tumor and normal prostate,bladder, skeletal muscle and colon. Increased expression of 10-H10 wasseen in prostate tumor and normal prostate, bladder, lung, colon, brainand large intestine. Increased expression of 9-B1 was seen in prostatetumor, breast tumor, and normal prostate, salivary gland, largeintestine and skin, with increased expression of 11 -C8 being seen inprostate tumor, and normal prostate and large intestine.

An additional cDNA fragment derived from the PCR-based normal prostatesubtraction, described above, was found to be prostate specific by bothmicro-array technology and RT-PCR. The determined cDNA sequence of thisclone (referred to as 9-A11) is provided in SEQ ID NO: 226. Comparisonof this sequence with those in the public databases revealed 99%identity to the known gene HOXB13.

Further studies led to the isolation of the clones 8-C6 and 8-H7. Thedetermined cDNA sequences for these clones are provided in SEQ ID NO:227 and 228, respectively. These sequences were found to show somehomology to previously isolated ESTs.

PCR and hybridization-based methodologies were employed to obtain longercDNA sequences for clone P20 (also referred to as P703P), yielding threeadditional cDNA fragments that progressively extend the 5′ end of thegene. These fragments, referred to as P703PDE5, P703P6.26, and P703PX-23(SEQ ID NO: 326, 328 and 330, with the predicted corresponding aminoacid sequences being provided in SEQ ID NO: 327, 329 and 331,respectively) contain additional 5′ sequence. P703PDE5 was recovered byscreening of a cDNA library (#141-26) with a portion of P703P as aprobe. P703P6.26 was recovered from a mixture of three prostate tumorcDNAs and P703PX_(—)23 was recovered from cDNA library (#438-48).Together, the additional sequences include all of the putative matureserine protease along with part of the putative signal sequence. Thefull-length cDNA sequence for P703P is provided in SEQ ID NO: 524, withthe corresponding amino acid sequence being provided in SEQ ID NO: 525.

P703P was found to show some homology to previously identifiedproteases, such as thrombin. The thrombin receptor has been shown to bepreferentially expressed in highly metastatic breast carcinoma cells andbreast carcinoma biopsy samples. Introduction of thrombin receptorantisense cDNA has been shown to inhibit the invasion of metastaticbreast carcinoma cells in culture. Antibodies against thrombin receptorinhibit thrombin receptor activation and thrombin-induced plateletactivation. Furthermore, peptides that resemble the receptor's tetheredligand domain inhibit platelet aggregation by thrombin. P703P may play arole in prostate cancer through a protease-activated receptor on thecancer cell or on stromal cells. The potential trypsin-like proteaseactivity of P703P may either activate a protease-activated receptor onthe cancer cell membrane to promote tumorgenesis or activate aprotease-activated receptor on the adjacent cells (such as stromalcells) to secrete growth factors and/or proteases (such as matrixmetalloproteinases) that could promote tumor angiogenesis, invasion andmetastasis. P703P may thus promote tumor progression and/or metastasisthrough the activation of protease-activated receptor. Polypeptides andantibodies that block the P703P-receptor interaction may therefore beusefully employed in the treatment of prostate cancer.

To determine whether P703P expression increases with increased severityof Gleason grade, an indicator of tumor stage, quantitative PCR analysiswas performed on prostate tumor samples with a range of Gleason scoresfrom 5 to >8. The mean level of P703P expression increased withincreasing Gleason score, indicating that P703P expression may correlatewith increased disease severity.

Further studies using a PCR-based subtraction library of a prostatetumor pool subtracted against a pool of normal tissues (referred to asJP: PCR subtraction) resulted in the isolation of thirteen additionalclones, seven of which did not share any significant homology to knownGenBank sequences. The determined cDNA sequences for these seven clones(P711P, P712P, novel 23, P774P, P775P, P710P and P768P) are provided inSEQ ID NO: 307-311, 313 and 315, respectively. The remaining six clones(SEQ ID NO: 316 and 321-325) were shown to share some homology to knowngenes. By microarray analysis, all thirteen clones showed three or morefold over-expression in prostate tissues, including prostate tumors, BPHand normal prostate as compared to normal non-prostate tissues. ClonesP711P, P712P, novel 23 and P768P showed over-expression in most prostatetumors and BPH tissues tested (n=29), and in the majority of normalprostate tissues (n=4), but background to low expression levels in allnormal tissues. Clones P774P, P775P and P710P showed comparatively lowerexpression and expression in fewer prostate tumors and BPH samples, withnegative to low expression in normal prostate.

Further studies led to the isolation of an extended cDNA sequence forP712P (SEQ ID NO: 552). The amino acid sequences encoded by 16 predictedopen reading frames present within the sequence of SEQ ID NO: 552 areprovided in SEQ ID NO: 553-568.

The full-length cDNA for P711 P was obtained by employing the partialsequence of SEQ ID NO: 307 to screen a prostate cDNA library.Specifically, a directionally cloned prostate cDNA library was preparedusing standard techniques. One million colonies of this library wereplated onto LB/Amp plates. Nylon membrane filters were used to liftthese colonies, and the cDNAs which were picked up by these filters weredenatured and cross-linked to the filters by UV light. The P711P cDNAfragment of SEQ ID NO: 307 was radio-labeled and used to hybridize withthese filters. Positive clones were selected, and cDNAs were preparedand sequenced using an automatic Perkin Elmer/Applied Biosystemssequencer. The determined full-length sequence of P711P is provided inSEQ ID NO: 382, with the corresponding predicted amino acid sequencebeing provided in SEQ ID NO: 383.

Using PCR and hybridization-based methodologies, additional cDNAsequence information was derived for two clones described above, 11-C9and 9-F3, herein after referred to as P707P and P714P, respectively (SEQID NO: 333 and 334). After comparison with the most recent GenBank,P707P was found to be a splice variant of the known gene HoxB13. Incontrast, no significant homologies to P714P were found. Further studiesemploying the sequence of SEQ ID NO: 334 as a probe in standardfull-length cloning methods, resulted in an extended cDNA sequence forP714P. This sequence is provided in SEQ ID NO: 698. This sequence wasfound to show some homology to the gene that encodes human ribosomalL23A protein.

Clones 8-B3, P89, P98, P130 and P201 (as disclosed in U.S. patentapplication Ser. No. 09/020,956, filed Feb. 9, 1998) were found to becontained within one contiguous sequence, referred to as P705P (SEQ IDNO: 335, with the predicted amino acid sequence provided in SEQ ID NO:336), which was determined to be a splice variant of the known gene NKX3.1.

Further studies on P775P resulted in the isolation of four additionalsequences (SEQ ID NO: 473-476) which are all splice variants of theP775P gene. The sequence of SEQ ID NO: 474 was found to contain two openreading frames (ORFs). The predicted amino acid sequences encoded bythese ORFs are provided in SEQ ID NO: 477 and 478. The cDNA sequence ofSEQ ID NO: 475 was found to contain an ORF which encodes the amino acidsequence of SEQ ID NO: 479. The cDNA sequence of SEQ ID NO: 473 wasfound to contain four ORFs. The predicted amino acid sequences encodedby these ORFs are provided in SEQ ID NO: 480-483. Additional splicevariants of P775P are provided in SEQ ID NO: 593-597.

Subsequent studies led to the identification of a genomic region onchromosome 22q11.2, known as the Cat Eye Syndrome region, that containsthe five prostate genes P704P, P712P, P774P, P775P and B305D. Therelative location of each of these five genes within the genomic regionis shown in FIG. 10. This region may therefore be associated withmalignant tumors, and other potential tumor genes may be containedwithin this region. These studies also led to the identification of apotential open reading frame (ORF) for P775P (provided in SEQ ID NO:533), which encodes the amino acid sequence of SEQ ID NO: 534.

Comparison of the clone of SEQ ID NO: 325 (referred to as P558S) withsequences in the GenBank and GeneSeq DNA databases showed that P558S isidentical to the prostate-specific transglutaminase gene, which is knownto have two forms. The full-length sequences for the two forms areprovided in SEQ ID NO: 773 and 774, with the corresponding amino acidsequences being provided in SEQ ID NO: 775 and 776, respectively. ThecDNA sequence of SEQ ID NO: 774 has a 15 pair base insert, resulting ina 5 amino acid insert in the corresponding amino acid sequence (SEQ IDNO: 776). This insert is not present in the sequence of SEQ ID NO: 773.

EXAMPLE 4 Synthesis of Polypeptides

Polypeptides may be synthesized on a Perkin Elmer/Applied Biosystems430A peptide synthesizer using FMOC chemistry with HPTU(O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate)activation. A Gly-Cys-Gly sequence may be attached to the amino terminusof the peptide to provide a method of conjugation, binding to animmobilized surface, or labeling of the peptide. Cleavage of thepeptides from the solid support may be carried out using the followingcleavage mixture: trifluoroaceticacid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleavingfor 2 hours, the peptides may be precipitated in coldmethyl-t-butyl-ether. The peptide pellets may then be dissolved in watercontaining 0.1% trifluoroacetic acid (TFA) and lyophilized prior topurification by C18 reverse phase HPLC. A gradient of 0%-60%acetonitrile (containing 0.1% TFA) in water (containing 0.1 % TFA) maybe used to elute the peptides. Following lyophilization of the purefractions, the peptides may be characterized using electrospray or othertypes of mass spectrometry and by amino acid analysis.

EXAMPLE 5 Further Isolation and Characterization of Prostate-specificPolypeptides by Pcr-based Subtraction

A cDNA library generated from prostate primary tumor mRNA as describedabove was subtracted with cDNA from normal prostate. The subtraction wasperformed using a PCR-based protocol (Clontech), which was modified togenerate larger fragments. Within this protocol, tester and driverdouble stranded cDNA were separately digested with five restrictionenzymes that recognize six-nucleotide restriction sites (MluI, MscI,PvuII, SalI and StuI). This digestion resulted in an average cDNA sizeof 600 bp, rather than the average size of 300 bp that results fromdigestion with RsaI according to the Clontech protocol. Thismodification did not affect the subtraction efficiency. Two testerpopulations were then created with different adapters, and the driverlibrary remained without adapters.

The tester and driver libraries were then hybridized using excess drivercDNA. In the first hybridization step, driver was separately hybridizedwith each of the two tester cDNA populations. This resulted inpopulations of (a) unhybridized tester cDNAs, (b) tester cDNAshybridized to other tester cDNAs, (c) tester cDNAs hybridized to drivercDNAs and (d) unhybridized driver cDNAs. The two separate hybridizationreactions were then combined, and rehybridized in the presence ofadditional denatured driver cDNA. Following this second hybridization,in addition to populations (a) through (d), a fifth population (e) wasgenerated in which tester cDNA with one adapter hybridized to testercDNA with the second adapter. Accordingly, the second hybridization stepresulted in enrichment of differentially expressed sequences which couldbe used as templates for PCR amplification with adaptor-specificprimers.

The ends were then filled in, and PCR amplification was performed usingadaptor-specific primers. Only population (e), which contained testercDNA that did not hybridize to driver cDNA, was amplified exponentially.A second PCR amplification step was then performed, to reduce backgroundand further enrich differentially expressed sequences.

This PCR-based subtraction technique normalizes differentially expressedcDNAs so that rare transcripts that are overexpressed in prostate tumortissue may be recoverable. Such transcripts would be difficult torecover by traditional subtraction methods.

In addition to genes known to be overexpressed in prostate tumor,seventy-seven further clones were identified. Sequences of these partialcDNAs are provided in SEQ ID NO: 29 to 305. Most of these clones had nosignificant homology to database sequences. Exceptions were JPTPN23 (SEQID NO: 231; similarity to pig valosin-containing protein), JPTPN30 (SEQID NO: 234; similarity to rat mRNA for proteasome subunit), JPTPN45 (SEQID NO: 243; similarity to rat norvegicus cytosolic NADP-dependentisocitrate dehydrogenase), JPTPN46 (SEQ ID NO: 244; similarity to humansubdlone H8 4 d4 DNA sequence), JP1D6 (SEQ ID NO: 265; similarity to G.gallus dynein light chain-A), JP8D6 (SEQ ID NO: 288; similarity to humanBAC clone RG016J04), JP8F5 (SEQ ID NO: 289; similarity to human subcloneH8 3 b5 DNA sequence), and JP8E9 (SEQ ID NO: 299; similarity to humanAlu sequence).

Additional studies using the PCR-based subtraction library consisting ofa prostate tumor pool subtracted against a normal prostate pool(referred to as PT-PN PCR subtraction) yielded three additional clones.Comparison of the cDNA sequences of these clones with the most recentrelease of GenBank revealed no significant homologies to the two clonesreferred to as P715P and P767P (SEQ ID NO: 312 and 314). The remainingclone was found to show some homology to the known gene KIAA0056 (SEQ IDNO: 318). Using microarray analysis to measure mRNA expression levels invarious tissues, all three clones were found to be over-expressed inprostate tumors and BPH tissues. Specifically, clone P715P wasover-expressed in most prostate tumors and BPH tissues by a factor ofthree or greater, with elevated expression seen in the majority ofnormal prostate samples and in fetal tissue, but negative to lowexpression in all other normal tissues. Clone P767P was over-expressedin several prostate tumors and BPH tissues, with moderate expressionlevels in half of the normal prostate samples, and background to lowexpression in all other normal tissues tested.

Further analysis, by microarray as described above, of the PT-PN PCRsubtraction library and of a DNA subtraction library containing cDNAfrom prostate tumor subtracted with a pool of normal tissue cDNAs, ledto the isolation of 27 additional clones (SEQ ID NO: 340-365 and 381)which were determined to be over-expressed in prostate tumor. The clonesof SEQ ID NO: 341, 342, 345, 347, 348, 349, 351, 355-359, 361, 362 and364 were also found to be expressed in normal prostate. Expression ofall 26 clones in a variety of normal tissues was found to be low orundetectable, with the exception of P544S (SEQ ID NO: 356) which wasfound to be expressed in small intestine. Of the 26 clones, 11 (SEQ IDNO: 340-349 and 362) were found to show some homology to previouslyidentified sequences. No significant homologies were found to the clonesof SEQ ID NO: 350, 351, 353-361, and 363-365.

Comparison of the sequence of SEQ ID NO: 362 with sequences in theGenBank and GeneSeq DNA databases showed that this clone (referred to asP788P) is identical to GeneSeq Accession No. X27262, which encodes aprotein found in the GeneSeq protein Accession No. Y00931. The fulllength cDNA sequence of P788P is shown in FIG. 12A (SEQ ID NO: 777),with the corresponding predicted amino acid being shown in FIG. 12B (SEQID NO: 778). Subsequently, a full-length cDNA sequence for P788P thatcontains polymorphisms not found in the sequence of SEQ ID NO: 779, wascloned multiple times by PCR amplification from cDNA prepared fromseveral RNA templates from three individuals. This determined cDNAsequence of this polymorphic variant of P788P is provided in SEQ ID NO:779, with the corresponding amino acid sequence being provided in SEQ IDNO: 780. The sequence of SEQ ID NO: 780 differs from that of SEQ ID NO:778 by six amino acid residues. The P788P protein has 7 potentialtransmembrane domains at the C-terminal portion and is predicted to be aplasma membrane protein with an extracellular N-terminal region.

Further studies on the clone of SEQ ID NO: 352 (referred to as P790P)led to the isolation of the full-length cDNA sequence of SEQ ID NO: 526.The corresponding predicted amino acid is provided in SEQ ID NO: 527.Data from two quantitative PCR experiments indicated that P790P isover-expressed in 11/15 tested prostate tumor samples and is expressedat low levels in spinal cord, with no expression being seen in all othernormal samples tested. Data from further PCR experiments and microarrayexperiments showed over-expression in normal prostate and prostate tumorwith little or no expression in other tissues tested. P790P wassubsequently found to show significant homology to a previouslyidentified G-protein coupled prostate tissue receptor.

Additional studies on the clone of SEQ ID NO: 354 (referred to as P776P)led to the isolation of an extended cDNA sequence, provided in SEQ IDNO: 569. The determined cDNA sequences of three additional splicevariants of P776P are provided in SEQ ID NO: 570-572. The amino acidsequences encoded by two predicted open reading frames (ORFs) containedwithin SEQ ID NO: 570, one predicted ORF contained within SEQ ID NO:571, and 11 predicted ORFs contained within SEQ ID NO: 569, are providedin SEQ ID NO: 573-586, respectively.

Comparison of the cDNA sequences for the clones P767P (SEQ ID NO: 314)and P777P (SEQ ID NO: 350) with sequences in the GenBank human ESTdatabase showed that the two clones matched many EST sequences incommon, suggesting that P767P and P777P may represent the same gene. ADNA consensus sequence derived from a DNA sequence alignment of P767P,P777P and multiple EST clones is provided in SEQ ID NO: 587. The aminoacid sequences encoded by three putative ORFs located within SEQ ID NO:587 are provided in SEQ ID NO: 588-590.

EXAMPLE 6 Peptide Priming of Mice and Propagation of CTL Lines

6.1. This Example illustrates the Preparation of a CTL Cell LineSpecific for Cells Expressing the P502S Gene.

Mice expressing the transgene for human HLA A2Kb (provided by Dr L.Sherman, The Scripps Research Institute, La Jolla, Calif.) wereimmunized with P2S#12 peptide (VLGWVAEL; SEQ ID NO: 306), which isderived from the P502S gene (also referred to herein as J1-17, SEQ IDNO: 8), as described by Theobald et al., Proc. Natl. Acad. Sci. USA92:11993-11997, 1995 with the following modifications. Mice wereimmunized with 100 μg of P2S#12 and 120 μg of an I-A^(b) binding peptidederived from hepatitis B Virus protein emulsified in incomplete Freund'sadjuvant. Three weeks later these mice were sacrificed and using a nylonmesh single cell suspensions prepared. Cells were then resuspended at6×10⁶ cells/ml in complete media (RPMI-1640; Gibco BRL, Gaithersburg,Md.) containing 10% FCS, 2 mM Glutamine (Gibco BRL), sodium pyruvate(Gibco BRL), non-essential amino acids (Gibco BRL), 2×10⁻⁵ M2-mercaptoethanol, 50 U/ml penicillin and streptomycin, and cultured inthe presence of irradiated (3000 rads) P2S#12-pulsed (5 mg/ml P2S#12 and10 mg/ml β2-microglobulin) LPS blasts (A2 transgenic spleens cellscultured in the presence of 7 μg/ml dextran sulfate and 25 μg/ml LPS for3 days). Six days later, cells (5×10⁵/ml) were restimulated with2.5×10⁶/ml peptide pulsed irradiated (20,000 rads) EL4A2Kb cells(Sherman et al, Science 258:815-818, 1992) and 3×10⁶/ml A2 transgenicspleen feeder cells. Cells were cultured in the presence of 20 U/mlIL-2. Cells continued to be restimulated on a weekly basis as described,in preparation for cloning the line.

P2S#12 line was cloned by limiting dilution analysis with peptide pulsedEL4 A2Kb tumor cells (1×10⁴ cells/well) as stimulators and A2 transgenicspleen cells as feeders (5×10⁵ cells/well) grown in the presence of 30U/ml IL-2. On day 14, cells were restimulated as before. On day 21,clones that were growing were isolated and maintained in culture.Several of these clones demonstrated significantly higher reactivity(lysis) against human fibroblasts (HLA A2Kb expressing) transduced withP502S than against control fibroblasts. An example is presented in FIG.1.

This data indicates that P2S #12 represents a naturally processedepitope of the P502S protein that is expressed in the context of thehuman HLA A2Kb molecule.

6.2. This Example Illustrates the Preparation of Murine CTL Lines andCTL Clones Specific for Cells Expressing the P501S Gene.

This series of experiments were performed similarly to that describedabove. Mice were immunized with the P1S#10 peptide (SEQ ID NO: 337),which is derived from the P501S gene (also referred to herein as L1-12,SEQ ID NO: 110). The P1S#10 peptide was derived by analysis of thepredicted polypeptide sequence for P501S for potential HLA-A2 bindingsequences as defined by published HLA-A2 binding motifs (Parker, K C.,et al, J. Immunol., 152:163, 1994). P1S#10 peptide was synthesized asdescribed in Example 4, and empirically tested for HLA-A2 binding usinga T cell based competition assay. Predicted A2 binding peptides weretested for their ability to compete HLA-A2 specific peptide presentationto an HLA-A2 restricted CTL clone (D150M58), which is specific for theHLA-A2 binding influenza matrix peptide fluM58. D150M58 CTL secretes TNFin response to self-presentation of peptide fluM58. In the competitionassay, test peptides at 100-200 μg/ml were added to cultures of D150M58CTL in order to bind HLA-A2 on the CTL. After thirty minutes, CTLcultured with test peptides, or control peptides, were tested for theirantigen dose response to the fluM58 peptide in a standard TNF bioassay.As shown in FIG. 3, peptide P1S#10 competes HLA-A2 restrictedpresentation of fluM58, demonstrating that peptide P1S#10 binds HLA-A2.

Mice expressing the transgene for human HLA A2Kb were immunized asdescribed by Theobald et al. (Proc. Natl. Acad. Sci. USA 92:11993-11997,1995) with the following modifications. Mice were immunized with 62.5 μgof P1S #10 and 120 μg of an I-A^(b) binding peptide derived fromHepatitis B Virus protein emulsified in incomplete Freund's adjuvant.Three weeks later these mice were sacrificed and single cell suspensionsprepared using a nylon mesh. Cells were then resuspended at 6×10⁶cells/ml in complete media (as described above) and cultured in thepresence of irradiated (3000 rads) P1S#10-pulsed (2 μg/ml P1S#10 and 10mg/ml β2-microglobulin) LPS blasts (A2 transgenic spleens cells culturedin the presence of 7 μg/ml dextran sulfate and 25 μg/ml LPS for 3 days).Six days later cells (5×10⁵/ml) were restimulated with 2.5×10⁶/mlpeptide-pulsed irradiated (20,000 rads) EL4A2Kb cells, as describedabove, and 3×10⁶/ml A2 transgenic spleen feeder cells. Cells werecultured in the presence of 20 U/ml IL-2. Cells were restimulated on aweekly basis in preparation for cloning. After three rounds of in vitrostimulations, one line was generated that recognized P1S#10-pulsedJurkat A2Kb targets and P501S-transduced Jurkat targets as shown in FIG.4.

A P1S#10-specific CTL line was cloned by limiting dilution analysis withpeptide pulsed EL4 A2Kb tumor cells (1×10⁴ cells/well) as stimulatorsand A2 transgenic spleen cells as feeders (5×10⁵ cells/well) grown inthe presence of 30 U/ml IL-2. On day 14, cells were restimulated asbefore. On day 21, viable clones were isolated and maintained inculture. As shown in FIG. 5, five of these clones demonstrated specificcytolytic reactivity against P501S-transduced Jurkat A2Kb targets. Thisdata indicates that P1S#10 represents a naturally processed epitope ofthe P501S protein that is expressed in the context of the human HLA-A2.1molecule.

EXAMPLE 7 Priming of CTL IN VIVO Using Naked DNA Immunization with aProstate Antigen

The prostate-specific antigen L1-12, as described above, is alsoreferred to as P501S. HLA A2Kb Tg mice (provided by Dr L. Sherman, TheScripps Research Institute, La Jolla, Calif.) were immunized with 100 μgP501S in the vector VR1012 either intramuscularly or intradermally. Themice were immunized three times, with a two week interval betweenimmunizations. Two weeks after the last immunization, immune spleencells were cultured with Jurkat A2Kb-P501S transduced stimulator cells.CTL lines were stimulated weekly. After two weeks of in vitrostimulation, CTL activity was assessed against P501S transduced targets.Two out of 8 mice developed strong anti-P501S CTL responses. Theseresults demonstrate that P501S contains at least one naturally processedHLA-A2-restricted CTL epitope.

EXAMPLE 8 Ability of Human T Cells to Recognize Prostate-specificPolypeptides

This Example illustrates the ability of T cells specific for a prostatetumor polypeptide to recognize human tumor.

Human CD8⁺ T cells were primed in vitro to the P2S-12 peptide (SEQ IDNO: 306) derived from P502S (also referred to as J1-17) using dendriticcells according to the protocol of Van Tsai et al. (Critical Reviews inImmunology 18:65-75, 1998). The resulting CD8⁺ T cell microcultures weretested for their ability to recognize the P2S-12 peptide presented byautologous fibroblasts or fibroblasts which were transduced to expressthe P502S gene in a γ-interferon ELISPOT assay (see Lalvani et al., J.Exp. Med. 186:859-865, 1997). Briefly, titrating numbers of T cells wereassayed in duplicate on 10⁴ fibroblasts in the presence of 3 μg/ml humanβ₂-microglobulin and 1 μg/ml P2S-12 peptide or control E75 peptide. Inaddition, T cells were simultaneously assayed on autologous fibroblaststransduced with the P502S gene or as a control, fibroblasts transducedwith HER-2/neu. Prior to the assay, the fibroblasts were treated with 10ng/ml γ-interferon for 48 hours to upregulate class I MHC expression.One of the microcultures (#5) demonstrated strong recognition of bothpeptide pulsed fibroblasts as well as transduced fibroblasts in aγ-interferon ELISPOT assay. FIG. 2A demonstrates that there was a strongincrease in the number of γ-interferon spots with increasing numbers ofT cells on fibroblasts pulsed with the P2S-12 peptide (solid bars) butnot with the control E75 peptide (open bars). This shows the ability ofthese T cells to specifically recognize the P2S-12 peptide. As shown inFIG. 2B, this microculture also demonstrated an increase in the numberof γ-interferon spots with increasing numbers of T cells on fibroblaststransduced to express the P502S gene but not the HER-2/neu gene. Theseresults provide additional confirmatory evidence that the P2S-12 peptideis a naturally processed epitope of the P502S protein. Furthermore, thisalso demonstrates that there exists in the human T cell repertoire, highaffinity T cells which are capable of recognizing this epitope. These Tcells should also be capable of recognizing human tumors which expressthe P502S gene.

EXAMPLE 9 Elicitation of Prostate Antigen-specific CTL Responses inHuman Blood

This Example illustrates the ability of a prostate-specific antigen toelicit a CTL response in blood of normal humans.

Autologous dendritic cells (DC) were differentiated from monocytecultures derived from PBMC of normal donors by growth for five days inRPMI medium containing 10% human serum, 50 ng/ml GMCSF and 30 ng/mlIL-4. Following culture, DC were infected overnight with recombinantP501S-expressing vaccinia virus at an M.O.I. of 5 and matured for 8hours by the addition of 2 micrograms/ml CD40 ligand. Virus wasinactivated by UV irradiation, CD8⁺ cells were isolated by positiveselection using magnetic beads, and priming cultures were initiated in24-well plates. Following five stimulation cycles using autologousfibroblasts retrovirally transduced to express P501S and CD80, CD8+lines were identified that specifically produced interferon-gamma whenstimulated with autologous P501S-transduced fibroblasts. TheP501S-specific activity of cell line 3A-1 could be maintained followingadditional stimulation cycles on autologous B-LCL transduced with P501S.Line 3A-1 was shown to specifically recognize autologous B-LCLtransduced to express P501S, but not EGFP-transduced autologous B-LCL,as measured by cytotoxicity assays (⁵¹Cr release) and interferon-gammaproduction (Interferon-gamma Elispot; see above and Lalvani et al., J.Exp. Med. 186:859-865, 1997). The results of these assays are presentedin FIGS. 6A and 6B.

EXAMPLE 10 Identification of a Naturally Processed CTL Epitope ContainedWithin a Prostate-specific Antigen

The 9-mer peptide p5 (SEQ ID NO: 338) was derived from the P703P antigen(also referred to as P20). The p5 peptide is immunogenic in human HLA-A2donors and is a naturally processed epitope. Antigen specific human CD8+T cells can be primed following repeated in vitro stimulations withmonocytes pulsed with p5 peptide. These CTL specifically recognizep5-pulsed and P703P-transduced target cells in both ELISPOT (asdescribed above) and chromium release assays. Additionally, immunizationof HLA-A2Kb transgenic mice with p5 leads to the generation of CTL lineswhich recognize a variety of HLA-A2Kb or HLA-A2 transduced target cellsexpressing P703P.

Initial studies demonstrating that p5 is a naturally processed epitopewere done using HLA-A2Kb transgenic mice. HLA-A2Kb transgenic mice wereimmunized subcutaneously in the footpad with 100 μg of p5 peptidetogether with 140 μg of hepatitis B virus core peptide (a Th peptide) inFreund's incomplete adjuvant. Three weeks post immunization, spleencells from immunized mice were stimulated in vitro with peptide-pulsedLPS blasts. CTL activity was assessed by chromium release assay fivedays after primary in vitro stimulation. Retrovirally transduced cellsexpressing the control antigen P703P and HLA-A2Kb were used as targets.CTL lines that specifically recognized both p5-pulsed targets as well asP703P-expressing targets were identified.

Human in vitro priming experiments demonstrated that the p5 peptide isimmunogenic in humans. Dendritic cells (DC) were differentiated frommonocyte cultures derived from PBMC of normal human donors by culturingfor five days in RPMI medium containing 10% human serum, 50 ng/ml humanGM-CSF and 30 ng/ml human IL-4. Following culture, the DC were pulsedwith 1 μg/ml. p5 peptide and cultured with CD8+ T cell enriched PBMC.CTL lines were restimulated on a weekly basis with p5-pulsed monocytes.Five to six weeks after initiation of the CTL cultures, CTL recognitionof p5-pulsed target cells was demonstrated. CTL were additionally shownto recognize human cells transduced to express P703P, demonstrating thatp5 is a naturally processed epitope.

Studies identifying a further peptide epitope (referred to as peptide 4)derived from the prostate tumor-specific antigen P703P that is capableof being recognized by CD4 T cells on the surface of cells in thecontext of HLA class II molecules were carried out as follows. The aminoacid sequence for peptide 4 is provided in SEQ ID NO: 781, with thecorresponding cDNA sequence being provided in SEQ ID NO: 782.

Twenty 15-mer peptides overlapping by 10 amino acids and derived fromthe carboxy-terminal fragment of P703P were generated using standardprocedures. Dendritic cells (DC) were derived from PBMC of a normalfemale donor using GM-CSF and IL-4 by standard protocols. CD4 T cellswere generated from the same donor as the DC using MACS beads andnegative selection. DC were pulsed overnight with pools of the 15-merpeptides, with each peptide at a final concentration of 0.25microgram/ml. Pulsed DC were washed and plated at 1×10⁴ cells/well of96-well V-bottom plates and purified CD4 T cells were added at1×10⁵/well. Cultures were supplemented with 60 ng/ml IL-6 and 10 ng/mlIL-12 and incubated at 37° C. Cultures were restimulated as above on aweekly basis using DC generated and pulsed as above as antigenpresenting cells, supplemented with 5 ng/ml IL-7 and 10 u/ml IL-2.Following 4 in vitro stimulation cycles, 96 lines (each linecorresponding to one well) were tested for specific proliferation andcytokine production in response to the stimulating pools with anirrelevant pool of peptides derived from mammaglobin being used as acontrol.

One line (referred to as 1-F9) was identified from pool #1 thatdemonstrated specific proliferation (measured by 3H proliferationassays) and cytokine production (measured by interferon-gamma ELISAassays) in response to pool #1 of P703P peptides. This line was furthertested for specific recognition of the peptide pool, specificrecognition of individual peptides in the pool, and in HLA mismatchanalyses to identify the relevant restricting allele. Line 1-F9 wasfound to specifically proliferate and produce interferon-gamma inresponse to peptide pool #1, and also to peptide 4 (SEQ ID NO: 781).Peptide 4 corresponds to amino acids 126-140 of SEQ ID NO: 327. Peptidetitration experiments were conducted to assess the sensitivity of line1-F9 for the specific peptide. The line was found to specificallyrespond to peptide 4 at concentrations as low as 0.25 ng/ml, indicatingthat the T cells are very sensitive and therefore likely to have highaffinity for the epitope.

To determine the HLA restriction of the P703P response, a panel ofantigen presenting cells (APC) was generated that was partially matchedwith the donor used to generate the T cells. The APC were pulsed withthe peptide and used in proliferation and cytokine assays together withline 1-F9. APC matched with the donor at HLA-DRB0701 and HLA-DQB02alleles were able to present the peptide to the T cells, indicating thatthe P703P-specific response is restricted to one of these alleles.

Antibody blocking assays were utilized to determine if the restrictingallele was HLA-DR0701 or HLA-DQ02. The anti-HLA-DR blocking antibodyL243 or an irrelevant isotype matched IgG2a were added to T cells andAPC cultures pulsed with the peptide RMPTVLQCVNVSVVS (SEQ ID NO: 781) at250 ng/ml. Standard interferon-gamma and proliferation assays wereperformed. Whereas the control antibody had no effect on the ability ofthe T cells to recognize peptide-pulsed APC, in both assays theanti-HLA-DR antibody completely blocked the ability of the T cells tospecifically recognize peptide-pulsed APC.

To determine if the peptide epitope RMPTVLQCVNVSVVS (SEQ ID NO: 781) wasnaturally processed, the ability of line 1-F9 to recognize APC pulsedwith recombinant P703P protein was examined. For these experiments anumber of recombinant P703P sources were utilized; E. coli-derivedP703P, Pichia-derived P703P and baculovirus-derived P703P. Irrelevantprotein controls used were E. coli-derived L3E a lung-specific antigen)and baculovirus-derived mammaglobin. In interferon-gamma ELISA assays,line 1-F9 was able to efficiently recognize both E. coli forms of P703Pas well as Pichia-derived recombinant P703P, while baculovirus-derivedP703P was recognized less efficiently. Subsequent Western blot analysisrevealed that the E coli and Pichia P703P protein preparations wereintact while the baculovirus P703P preparation was approximately 75%degraded. Thus, peptide RMPTVLQCVNVSVVS (SEQ ID NO: 781) from P703P is anaturally processed peptide epitope derived from P703P and presented toT cells in the context of HLA-DRB-0701

In further studies, twenty-four 15-mer peptides overlapping by 10 aminoacids and derived from the N-terminal fragment of P703P (correspondingto amino acids 27-154 of SEQ ID NO: 525) were generated by standardprocedures and their ability to be recognized by CD4 cells wasdetermined essentially as described above. DC were pulsed overnight withpools of the peptides with each peptide at a final concentration of 10microgram/ml. A large number of individual CD4 T cell lines (65/480)demonstrated significant proliferation and cytokine release (IFN-gamma)in response to the P703P peptide pools but not to a control peptidepool. The CD4 T cell lines which demonstrated specific activity wererestimulated on the appropriate pool of P703P peptides and reassayed onthe individual peptides of each pool as well as a peptide dose titrationof the pool of peptides in a IFN-gamma release assay and in aproliferation assay.

Sixteen immunogenic peptides were recognized by the T cells from theentire set of peptide antigens tested. The amino acid sequences of thesepeptides are provided in SEQ ID NO: 799-814, with the corresponding cDNAsequences being provided in SEQ ID NO: 783-798, respectively. In somecases the peptide reactivity of the T cell line could be mapped to asingle peptide, however some could be mapped to more than one peptide ineach pool. Those CD4 T cell lines that displayed a representativepattern of recognition from each peptide pool with a reasonable affinityfor peptide were chosen for further analysis (I-1A, -6A; II-4C, -5E;III-6E, IV-4B, -3F, -9B, -10F, V-5B, -4D, and -10F). These CD4 T cellslines were restimulated on the appropriate individual peptide andreassayed on autologous DC pulsed with a truncated form of recombinantP703P protein made in E. coli (a.a. 96-254 of SEQ ID NO: 525),full-length P703P made in the baculovirus expression system, and afusion between influenza virus NS1 and P703P made in E. coli. Of the Tcell lines tested, line I-1A recognized specifically the truncated formof P703P (E. coli) but no other recombinant form of P703P. This linealso recognized the peptide used to elicit the T cells. Line 2-4Crecognized the truncated form of P703P (E. coli) and the full lengthform of P703P made in baculovirus, as well as peptide. The remaining Tcell lines tested were either peptide-specific only (II-5E, II-6F,IV-4B, IV-3F, IV-9B, IV-10F, V-5B and V-4D) or were non-responsive toany antigen tested (V-10F). These results demonstrate that the peptidesequence RPLLANDLMLIKLDE (SEQ ID NO: 814; corresponding to a.a. 110-124of SEQ ID NO: 525) recognized by the T cell line I-1A, and the peptidesequences SVSESDTIRSISIAS (SEQ ID NO: 811; corresponding to a.a. 125-139of SEQ ID NO: 525) and ISIASQCPTAGNSCL (SEQ ID NO: 810; corresponding toa.a. 135-149 of SEQ ID NO: 525) recognized by the T cell line II-4C maybe naturally processed epitopes of the P703P protein.

EXAMPLE 11 Expression of a Breast Tumor-derived Antigen in Prostate

Isolation of the antigen B305D from breast tumor by differential displayis described in US patent application Ser. No. 08/700,014, filed Aug.20, 1996. Several different splice forms of this antigen were isolated.The determined cDNA sequences for these splice forms are provided in SEQID NO: 366-375, with the predicted amino acid sequences corresponding tothe sequences of SEQ ID NO: 292, 298 and 301-303 being provided in SEQID NO: 299-306, respectively. In further studies, a splice variant ofthe cDNA sequence of SEQ ID NO: 366 was isolated which was found tocontain an additional guanine residue at position 884 (SEQ ID NO: 530),leading to a frameshift in the open reading frame. The determined DNAsequence of this ORF is provided in SEQ ID NO: 531. This frameshiftgenerates a protein sequence (provided in SEQ ID NO: 532) of 293 aminoacids that contains the C-terminal domain common to the other isoformsof B305D but that differs in the N-terminal region.

The expression levels of B305D in a variety of tumor and normal tissueswere examined by real time PCR and by Northern analysis. The resultsindicated that B305D is highly expressed in breast tumor, prostatetumor, normal prostate and normal testes, with expression being low orundetectable in all other tissues examined (colon tumor, lung tumor,ovary tumor, and normal bone marrow, colon, kidney, liver, lung, ovary,skin, small intestine, stomach). Using real-time PCR on a panel ofprostate tumors, expression of B305D in prostate tumors was shown toincrease with increasing Gleason grade, demonstrating that expression ofB305D increases as prostate cancer progresses.

EXAMPLE 12 Generation of Human CTL IN VITRO Using Whole Gene Priming andStimulation Techniques with Prostate-specific Antigen

Using in vitro whole-gene priming with P501S-vaccinia infected DC (see,for example, Yee et al, The Journal of Immunology, 157(9):4079-86,1996), human CTL lines were derived that specifically recognizeautologous fibroblasts transduced with P501S (also known as L1-12), asdetermined by interferon-γ ELISPOT analysis as described above. Using apanel of HLA-mismatched B-LCL lines transduced with P501S, these CTLlines were shown to be likely restricted to HLAB class I allele.Specifically, dendritic cells (DC) were differentiated from monocytecultures derived from PBMC of normal human donors by growing for fivedays in RPMI medium containing 10% human serum, 50 ng/ml human GM-CSFand 30 ng/ml human IL-4. Following culture, DC were infected overnightwith recombinant P501S vaccinia virus at a multiplicity of infection(M.O.I) of five, and matured overnight by the addition of 3 μg/ml CD40ligand. Virus was inactivated by UV irradiation. CD8⁺ T cells wereisolated using a magnetic bead system, and priming cultures wereinitiated using standard culture techniques. Cultures were restimulatedevery 7-10 days using autologous primary fibroblasts retrovirallytransduced with P501S and CD80. Following four stimulation cycles, CD8+T cell lines were identified that specifically produced interferon-ywhen stimulated with P501S and CD80-transduced autologous fibroblasts. Apanel of HLA-mismatched B-LCL lines transduced with P501S were generatedto define the restriction allele of the response. By measuringinterferon-γ in an ELISPOT assay, the P501S specific response was shownto be likely restricted by HLA B alleles. These results demonstrate thata CD8+ CTL response to P501S can be elicited.

To identify the epitope(s) recognized, cDNA encoding P501S wasfragmented by various restriction digests, and sub-cloned into theretroviral expression vector pBIB-KS. Retroviral supernatants weregenerated by transfection of the helper packaging line Phoenix-Ampho.Supernatants were then used to transduce Jurkat/A2Kb cells for CTLscreening. CTL were screened in IFN-gamma ELISPOT assays against theseA2Kb targets transduced with the “library” of P501S fragments. Initialpositive fragments P501S/H3 and P501S/F2 were sequenced and found toencode amino acids 106-553 and amino acids 136-547, respectively, of SEQID NO: 113. A truncation of H3 was made to encode amino acid residues106-351 of SEQ ID NO: 113, which was unable to stimulate the CTL, thuslocalizing the epitope to amino acid residues 351-547. Additionalfragments encoding amino acids 1-472 (Fragment A) and amino acids 1-351(Fragment B) were also constructed. Fragment A but not Fragment Bstimulated the CTL thus localizing the epitope to amino acid residues351-472. Overlapping 20-mer and 18-mer peptides representing this regionwere tested by pulsing Jurkat/A2Kb cells versus CTL in an IFN-gammaassay. Only peptides P501S-369(20) and P501S-369(18) stimulated the CTL.Nine-mer and 10-mer peptides representing this region were synthesizedand similarly tested. Peptide P501S-370 (SEQ ID NO: 539) was the minimal9-mer giving a strong response. Peptide P501S-376 (SEQ ID NO: 540) alsogave a weak response, suggesting that it might represent across-reactive epitope.

In subsequent studies, the ability of primary human B cells transducedwith P501S to prime MHC class I-restricted, P501S-specific, autologousCD8 T cells was examined. Primary B cells were derived from PBMC of ahomozygous HLA-A2 donor by culture in CD40 ligand and IL-4, transducedat high frequency with recombinant P501S in the vector pBIB, andselected with blastocidin-S. For in vitro priming, purified CD8+ T cellswere cultured with autologous CD40 ligand+IL-4 derived, P501S-transducedB cells in a 96-well microculture formnat. These CTL microcultures werere-stimulated with P501S-transduced B cells and then assayed forspecificity. Following this initial screen, microcultures withsignificant signal above background were cloned on autologousEBV-transformed B cells (BLCL), also transduced with P501S. UsingIFN-gamma ELISPOT for detection, several of these CD8 T cell clones werefound to be specific for P501S, as demonstrated by reactivity toBLCL/P501S but not BLCL transduced with control antigen. It was furtherdemonstrated that the anti-P501S CD8 T cell specificity isHLA-A2-restricted. First, antibody blocking experiments withanti-HLA-A,B,C monoclonal antibody (W6.32), anti-HLA-B,C monoclonalantibody (B1.23.2) and a control monoclonal antibody showed that onlythe anti-HLA-A,B,C antibody blocked recognition of P501S-expressingautologous BLCL. Secondly, the anti-P501S CTL also recognized an HLA-A2matched, heterologous BLCL transduced with P501S, but not thecorresponding EGFP transduced control BLCL.

EXAMPLE 13 Identification of Prostate-specific Antigens by MicroarrayAnalysis

This Example describes the isolation of certain prostate-specificpolypeptides from a prostate tumor cDNA library.

A human prostate tumor cDNA expression library as described above wasscreened using microarray analysis to identify clones that display atleast a three fold over-expression in prostate tumor and/or normalprostate tissue, as compared to non-prostate normal tissues (notincluding testis). 372 clones were identified, and 319 were successfullysequenced. Table I presents a summary of these clones, which are shownin SEQ ID NOs:385-400. Of these sequences SEQ ID NOs:386, 389, 390 and392 correspond to novel genes, and SEQ ID NOs: 393 and 396 correspond topreviously identified sequences. The others (SEQ ID NOs:385, 387, 388,391, 394, 395 and 397-400) correspond to known sequences, as shown inTable I.

TABLE I Summary of Prostate Tumor Antigens Previously Identified KnownGenes Genes Novel Genes T-cell gamma chain P504S 23379 (SEQ ID NO: 389)Kallikrein P1000C 23399 (SEQ ID NO: 392) Vector P501S 23320 (SEQ ID NO:386) CGI-82 protein mRNA P503S 23381 (SEQ ID (23319; SEQ ID NO: NO: 390)385) PSA P510S Ald. 6 Dehyd. P784P L-iditol-2 dehydrogenase P502S(23376; SEQ ID NO: 388) Ets transcription factor P706P PDEF (22672; SEQID NO: 398) hTGR (22678; SEQ ID 19142.2, bangur. NO: 399) seq (22621;SEQ ID NO: 396) KIAA0295 (22685; SEQ 5566.1 Wang (23404; ID NO: 400) SEQID NO: 393) Prostatic Acid Phos- P712P phatase (22655; SEQ ID NO: 397)transglutaminase (22611; P778P SEQ ID NO: 395) HDLBP (23508; SEQ ID NO:394) CGI-69 Protein (23367; SEQ ID NO: 387) KIAA0122 (23383; SEQ ID NO:391) TEEG

CGI-82 showed 4.06 fold over-expression in prostate tissues as comparedto other normal tissues tested. It was over-expressed in 43% of prostatetumors, 25% normal prostate, not detected in other normal tissuestested. L-iditol-2 dehydrogenase showed 4.94 fold over-expression inprostate tissues as compared to other normal tissues tested. It wasover-expressed in 90% of prostate tumors, 100% of normal prostate, andnot detected in other normal tissues tested. Ets transcription factorPDEF showed 5.55 fold over-expression in prostate tissues as compared toother normal tissues tested. It was over-expressed in 47% prostatetumors, 25% normal prostate and not detected in other normal tissuestested. hTGR1 showed 9.11 fold over-expression in prostate tissues ascompared to other normal tissues tested. It was over-expressed in 63% ofprostate tumors and is not detected in normal tissues tested includingnormal prostate. KIAA0295 showed 5.59 fold over-expression in prostatetissues as compared to other normal tissues tested. It wasover-expressed in 47% of prostate tumors, low to undetectable in normaltissues tested including normal prostate tissues. Prostatic acidphosphatase showed 9.14 fold over-expression in prostate tissues ascompared to other normal tissues tested. It was over-expressed in 67% ofprostate tumors, 50% of normal prostate, and not detected in othernormal tissues tested. Transglutaminase showed 14.84 foldover-expression in prostate tissues as compared to other normal tissuestested. It was over-expressed in 30% of prostate tumors, 50% of normalprostate, and is not detected in other normal tissues tested. Highdensity lipoprotein binding protein (HDLBP) showed 28.06 foldover-expression in prostate tissues as compared to other normal tissuestested. It was over-expressed in 97% of prostate tumors, 75% of normalprostate, and is undetectable in all other normal tissues tested. CGI-69showed 3.56 fold over-expression in prostate tissues as compared toother normal tissues tested. It is a low abundant gene, detected in morethan 90% of prostate tumors, and in 75% normal prostate tissues. Theexpression of this gene in normal tissues was very low. KIAA0122 showed4.24 fold over-expression in prostate tissues as compared to othernormal tissues tested. It was over-expressed in 57% of prostate tumors,it was undetectable in all normal tissues tested including normalprostate tissues. 19142.2 bangur showed 23.25 fold over-expression inprostate tissues as compared to other normal tissues tested. It wasover-expressed in 97% of prostate tumors and 100% of normal prostate. Itwas undetectable in other normal tissues tested. 5566.1 Wang showed 3.31fold over-expression in prostate tissues as compared to other normaltissues tested. It was over-expressed in 97% of prostate tumors, 75%normal prostate and was also over-expressed in normal bone marrow,pancreas, and activated PBMC. Novel clone 23379 (also referred to asP553S) showed 4.86 fold over-expression in prostate tissues as comparedto other normal tissues tested. It was detectable in 97% of prostatetumors and 75% normal prostate and is undetectable in all other normaltissues tested. Novel clone 23399 showed 4.09 fold over-expression inprostate tissues as compared to other normal tissues tested. It wasover-expressed in 27% of prostate tumors and was undetectable in allnormal tissues tested including normal prostate tissues. Novel clone23320 showed 3.15 fold over-expression in prostate tissues as comparedto other normal tissues tested. It was detectable in all prostate tumorsand 50% of normal prostate tissues. It was also expressed in normalcolon and trachea. Other normal tissues do not express this gene at highlevel.

Subsequent full-length cloning studies on P553S, using standardtechniques, revealed that this clone is an incomplete spliced form ofP501S. The determined cDNA sequences for four splice variants of P553Sare provided in SEQ ID NO: 702-705. An amino acid sequence encoded bySEQ ID NO: 705 is provided in SEQ ID NO: 706. The cDNA sequence of SEQID NO: 702 was found to contain two open reading frames (ORFs). Theamino acid sequences encoded by these two ORFs are provided in SEQ IDNO: 707 and 708.

EXAMPLE 14 Identification of Prostate-specific Antigens by ElectronicSubtraction

This Example describes the use of an electronic subtraction technique toidentify prostate-specific antigens.

Potential prostate-specific genes present in the GenBank human ESTdatabase were identified by electronic subtraction (similar to thatdescribed by Vasmatizis et al., Proc. Natl. Acad. Sci. USA 95:300-304,1998). The sequences of EST clones (43,482) derived from variousprostate libraries were obtained from the GenBank public human ESTdatabase. Each prostate EST sequence was used as a query sequence in aBLASTN (National Center for Biotechnology Information) search againstthe human EST database. All matches considered identical (length ofmatching sequence >100 base pairs, density of identical matches overthis region >70%) were grouped (aligned) together in a cluster. Clusterscontaining more than 200 ESTs were discarded since they probablyrepresented repetitive elements or highly expressed genes such as thosefor ribosomal proteins. If two or more clusters shared common ESTs,those clusters were grouped together into a “supercluster,” resulting in4,345 prostate superclusters.

Records for the 479 human cDNA libraries represented in the GenBankrelease were downloaded to create a database of these cDNA libraryrecords. These 479 cDNA libraries were grouped into three groups: Plus(normal prostate and prostate tumor libraries, and breast cell linelibraries, in which expression was desired), Minus (libraries from othernormal adult tissues, in which expression was not desirable), and Other(libraries from fetal tissue, infant tissue, tissues found only inwomen, non-prostate tumors and cell lines other than prostate celllines, in which expression was considered to be irrelevant). A summaryof these library groups is presented in Table II.

TABLE II Prostate cDNA Libraries and ESTs Library # of Libraries # ofESTs Plus 25 43,482 Normal 11 18,875 Tumor 11 21,769 Cell lines 3 2,838Minus 166 Other 287

Each supercluster was analyzed in terms of the ESTs within thesupercluster. The tissue source of each EST clone was noted and used toclassify the superclusters into four groups: Type 1-EST clones found inthe Plus group libraries only; no expression detected in Minus or Othergroup libraries; Type 2-EST clones derived from the Plus and Other grouplibraries only; no expression detected in the Minus group; Type 3-ESTclones derived from the Plus, Minus and Other group libraries, but thenumber of ESTs derived from the Plus group is higher than in either theMinus or Other groups; and Type 4-EST clones derived from Plus, Minusand Other group libraries, but the number derived from the Plus group ishigher than the number derived from the Minus group. This analysisidentified 4,345 breast clusters (see Table III). From these clusters,3,172 EST clones were ordered from Research Genetics, Inc., and werereceived as frozen glycerol stocks in 96-well plates.

TABLE III Prostate Cluster Summary # of # of ESTs Type SuperclustersOrdered 1 688 677 2 2899 2484 3 85 11 4 673 0 Total 4345 3172

The EST clone inserts were PCR-amplified using amino-linked PCR primersfor Synteni microarray analysis. When more than one PCR product wasobtained for a particular clone, that PCR product was not used forexpression analysis. In total, 2,528 clones from the electronicsubtraction method were analyzed by microarray analysis to identifyelectronic subtraction breast clones that had high levels of tumor vs.normal tissue mRNA. Such screens were performed using a Synteni (PaloAlto, Calif.) microarray, according to the manufacturer's instructions(and essentially as described by Schena et al., Proc. Natl. Acad. Sci.USA 93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA94:2150-2155, 1997). Within these analyses, the clones were arrayed onthe chip, which was then probed with fluorescent probes generated fromnormal and tumor prostate cDNA, as well as various other normal tissues.The slides were scanned and the fluorescence intensity was measured.

Clones with an expression ratio greater than 3 (i.e., the level inprostate tumor and normal prostate mRNA was at least three times thelevel in other normal tissue mRNA) were identified as prostatetumor-specific sequences (Table IV). The sequences of these clones areprovided in SEQ ID NO: 401-453, with certain novel sequences shown inSEQ ID NO: 407, 413, 416-419, 422, 426, 427 and 450.

TABLE IV Prostate-tumor Specific Clones Sequence SEQ ID NO. DesignationComments 401 22545 previously identified P1000C 402 22547 previouslyidentified P704P 403 22548 known 404 22550 known 405 22551 PSA 406 22552prostate secretory protein 94 407 22553 novel 408 22558 previouslyidentified P509S 409 22562 glandular kallikrein 410 22565 previouslyidentified P1000C 411 22567 PAP 412 22568 B1006C (breast tumor antigen)413 22570 novel 414 22571 PSA 415 22572 previously identified P706P 41622573 novel 417 22574 novel 418 22575 novel 419 22580 novel 420 22581PAP 421 22582 prostatic secretory protein 94 422 22583 novel 423 22584prostatic secretory protein 94 424 22585 prostatic secretory protein 94425 22586 known 426 22587 novel 427 22588 novel 428 22589 PAP 429 22590known 430 22591 PSA 431 22592 known 432 22593 Previously identifiedP777P 433 22594 T cell receptor gamma chain 434 22595 Previouslyidentified P705P 435 22596 Previously identified P707P 436 22847 PAP 43722848 known 438 22849 prostatic secretory protein 57 439 22851 PAP 44022852 PAP 441 22853 PAP 442 22854 previously identified P509S 443 22855previously identified P705P 444 22856 previously identified P774P 44522857 PSA 446 23601 previously identified P777P 447 23602 PSA 448 23605PSA 449 23606 PSA 450 23612 novel 451 23614 PSA 452 23618 previouslyidentified P1000C 453 23622 previously identified P705P

Further studies on the clone of SEQ ID NO: 407 (also referred to asP1020C) led to the isolation of an extended cDNA sequence provided inSEQ ID NO: 591. This extended cDNA sequence was found to contain an openreading frame that encodes The predicted amino acid sequence of SEQ IDNO: 592. The P1020C cDNA and amino acid sequences were found to showsome similarity to the human endogenous retroviral HERV-K pol gene andprotein.

EXAMPLE 15 Further Identification of Prostate-specific Antigens byMicroarray Analysis

This Example describes the isolation of additional prostate-specificpolypeptides from a prostate tumor cDNA library.

A human prostate tumor cDNA expression library as described above wasscreened using microarray analysis to identify clones that display atleast a three fold over-expression in prostate tumor and/or normalprostate tissue, as compared to non-prostate normal tissues (notincluding testis). 142 clones were identified and sequenced. Certain ofthese clones are shown in SEQ ID NO: 454-467. Of these sequences, SEQ IDNO: 459-461 represent novel genes. The others (SEQ ID NO: 454-458 and461-467) correspond to known sequences.

EXAMPLE 16 Further Characterization of Prostate-specific Antigen P710P

This Example describes the full length cloning of P710P.

The prostate cDNA library described above was screened with the P710Pfragment described above. One million colonies were plated onLB/Ampicillin plates. Nylon membrane filters were used to lift thesecolonies, and the cDNAs picked up by these filters were then denaturedand cross-linked to the filters by UV light. The P710P fragment wasradiolabeled and used to hybridize with the filters. Positive cDNAclones were selected and their cDNAs recovered and sequenced by anautomatic Perkin Elmer/Applied Biosystems Division Sequencer. Foursequences were obtained, and are presented in SEQ ID NO: 468-471. Thesesequences appear to represent different splice variants of the P710Pgene. Subsequent comparison of the cDNA sequences of P710P with those inGenbank releaved homology to the DD3 gene (Genbank accession numbersAF103907 & AF103908). The cDNA sequence of DD3 is provided in SEQ ID NO:690.

EXAMPLE 17 Protein Expression of Prostate-specific Antigens

This example describes the expression and purification ofprostate-specific antigens in E. coli, baculovirus and mammalian cells.

A) Expression of P501S in E. Coli

Expression of the full-length form of P501S was attempted by firstcloning P501S without the leader sequence (amino acids 36-553 of SEQ IDNO: 113) downstream of the first 30 amino acids of the M. tuberculosisantigen Ra12 (SEQ ID NO: 484) in pET17b. Specifically, P501S DNA wasused to perform PCR using the primers AW025 (SEQ ID NO: 485) and AW003(SEQ ID NO: 486). AW025 is a sense cloning primer that contains aHindIII site. AW003 is an antisense cloning primer that contains anEcoRI site. DNA amplification was performed using 5 μl 10×Pfu buffer, 1μl 20 mM dNTPs, 1 μleach of the PCR primers at 10 μM concentration, 40μl water, 1 μPfu DNA polymerase (Stratagene, La Jolla, Calif.) and 1 μlDNA at 100 ng/μl. Denaturation at 95° C. was performed for 30 sec,followed by 10 cycles of 95° C. for 30 sec, 60° C. for 1 min and by 72°C. for 3 min. 20 cycles of 95° C. for 30 sec, 65° C. for 1 min and by72° C. lastly by 1 cycle of 72° C. for 10 min. The PCR product wascloned to Ra12m/pET17b using HindIII and EcoRI. The sequence of theresulting fusion construct (referred to as Ra12-P501S-F) was confirmedby DNA sequencing.

The fusion construct was transformed into BL21(DE3)pLysE, pLysS andCodonPlus E. coli (Stratagene) and grown overnight in LB broth withkanamycin. The resulting culture was induced with IPTG. Protein wastransferred to PVDF membrane and blocked with 5% non-fat milk (inPBS-Tween buffer), washed three times and incubated with mouse anti-Histag antibody (Clontech) for 1 hour. The membrane was washed 3 times andprobed with HRP-Protein A (Zymed) for 30 min. Finally, the membrane waswashed 3 times and developed with ECL (Amersham). No expression wasdetected by Western blot. Similarly, no expression was detected byWestern blot when the Ra12-P501S-F fusion was used for expression inBL2ICodonPlus by CE6 phage (Invitrogen).

An N-terminal fragment of P501S (amino acids 36-325 of SEQ ID NO: 113)was cloned down-stream of the first 30 amino acids of the M.tuberculosis antigen Ra12 in pET1 7b as follows. P501S DNA was used toperform PCR using the primers AW025 (SEQ ID NO: 485) and AW027 (SEQ IDNO: 487). AW027 is an antisense cloning primer that contains an EcoRIsite and a stop codon. DNA amplification was performed essentially asdescribed above. The resulting PCR product was cloned to Ra12 in pET17bat the HindIII and EcoRI sites. The fusion construct (referred to asRa12-P501S-N) was confirmed by DNA sequencing.

The Ra12-P501S-N fusion construct was used for expression inBL21(DE3)pLysE, pLysS and CodonPlus, essentially as described above.Using Western blot analysis, protein bands were observed at the expectedmolecular weight of 36 kDa. Some high molecular weight bands were alsoobserved, probably due to aggregation of the recombinant protein. Noexpression was detected by Western blot when the Ra12-PSO51S-F fusionwas used for expression in BL21 CodonPlus by CE6 phage.

A fusion construct comprising a C-terminal portion of P501S (amino acids257-553 of SEQ ID NO: 113) located down-stream of the first 30 aminoacids of the M. tuberculosis antigen Ra12 (SEQ ID NO: 484) was preparedas follows. P501S DNA was used to perform PCR using the primers AW026(SEQ ID NO: 488) and AW003 (SEQ ID NO: 486). AW026 is a sense cloningprimer that contains a HindIII site. DNA amplification was performedessentially as described above. The resulting PCR product was cloned toRa12 in pET17b at the HindIII and EcoRI sites. The sequence for thefusion construct (referred to as Ra12-P501S-C) was confirmed.

The Ra12-P501S-C fusion construct was used for expression inBL21(DE3)pLysE, pLysS and CodonPlus, as described above. A small amountof protein was detected by Western blot, with some molecular weightaggregates also being observed. Expression was also detected by Westernblot when the Ra12-P501S-C fusion was used for expression in BL21CodonPlus induced by CE6 phage.

A fusion construct comprising a fragment of P501S (amino acids 36-298 ofSEQ ID NO: 113) located down-stream of the M. tuberculosis antigen Ra12(SEQ ID NO: 848) was prepared as follows. P501S DNA was used to performPCR using the primers AW042 (SEQ ID NO: 849) and AW053 (SEQ ID NO: 850).AW042 is a sense cloning primer that contains a EcoRI site. AW053 is anantisense primer with stop and Xho I sites. DNA amplification wasperformed essentially as described above. The resulting PCR product wascloned to Ra12 in pET17b at the EcoRI and Xho I sites. The resultingfusion construct (referred to as Ra12-P501S-E2) was expressed in B834(DE3) pLys S E. coli host cells in TB media for 2 h at room temperature.Expressed protein was purified by washing the inclusion bodies andrunning on a Ni-NTA column. The purified protein stayed soluble inbuffer containing 20 mM Tris-HCl (pH 8), 100 mM NaCl, 10 mM β-Me and 5%glycerol. The determined cDNA and amino acid sequences for the expressedfusion protein are provided in SEQ ID NO: 851 and 852, respectfully.

B) Expression of P501S in Baculovirus

The Bac-to-Bac baculovirus expression system (BRL Life Technologies,Inc.) was used to express P501S protein in insect cells. Full-lengthP501S (SEQ ID NO: 113) was amplified by PCR and cloned into the XbaIsite of the donor plasmid pFastBacl. The recombinant bacmid andbaculovirus were prepared according to the manufacturer's instructions.The recombinant baculovirus was amplified in Sf9 cells and the hightiter viral stocks were utilized to infect High Five cells (Invitrogen)to make the recombinant protein. The identity of the full-length proteinwas confirmed by N-terminal sequencing of the recombinant protein and byWestern blot analysis (FIG. 7). Specifically, 0.6 million High Fivecells in 6-well plates were infected with either the unrelated controlvirus BV/ECD_PD (lane 2), with recombinant baculovirus for P501S atdifferent amounts or MOIs (lanes 4-8), or were uninfected (lane 3). Celllysates were run on SDS-PAGE under educing conditions and analyzed byWestern blot with the anti-P501S monoclonal antibody P501S-10E3-G4D3(prepared as described below). Lane 1 is the biotinylated roteinmolecular weight marker (BioLabs).

The localization of recombinant P501S in the insect cells wasinvestigated as follows. The insect cells overexpressing P501S werefractionated into fractions of nucleus, mitochondria, membrane andcytosol. Equal amounts of protein from each fraction were analyzed byWestern blot with a monoclonal antibody against P501S. Due to the schemeof fractionation, both nucleus and mitochondria fractions contain someplasma membrane components. However, the membrane fraction is basicallyfree from mitochondria and nucleus. P501S was found to be present in allfractions that contain the membrane component, suggesting that P501S maybe associated with plasma membrane of the insect cells expressing therecombinant protein.

C) Expression of P501S in Mammalian Cells

Full-length P501S (553AA) was cloned into various mammalian expressionvectors, including pCEP4 (Invitrogen), pVR1012 (Vical, San Diego,Calif.) and a modified form of the retroviral vector pBMN, referred toas pBIB. Transfection of P501S/pCEP4 and P501S/pVR1012 into HEK293fibroblasts was carried out using the Fugene transfection reagent(Boehringer Mannheim). Briefly, 2 μl of Fugene reagent was diluted into100 μl of serum-free media and incubated at room temperature for 5-10min. This mixture was added to 1 μg of P501S plasmid DNA, mixed brieflyand incubated for 30 minutes at room temperature. The Fugene/DNA mixturewas added to cells and incubated for 24-48 hours. Expression ofrecombinant P501S in transfected HEK293 fibroblasts was detected bymeans of Western blot employing a monoclonal antibody to P501S.

Transfection of p501S/pCEP4 into CHO-K cells (American Type CultureCollection, Rockville, Md.) was carried out using GenePortertransfection reagent (Gene Therapy Systems, San Diego, Calif.). Briefly,15 μl of GenePorter was diluted in 500 μl of serum-free media andincubated at room temperature for 10 min. The GenePorter/media mixturewas added to 2 μg of plasmid DNA that was diluted in 500 μl ofserum-free media, mixed briefly and incubated for 30 min at roomtemperature. CHO-K cells were rinsed in PBS to remove serum proteins,and the GenePorter/DNA mix was added and incubated for 5 hours. Thetransfected cells were then fed an equal volume of 2×media and incubatedfor 24-48 hours.

FACS analysis of P501S transiently infected CHO-K cells, demonstratedsurface expression of P501S. Expression was detected using rabbitpolyclonal antisera raised against a P501S peptide, as described below.Flow cytometric analysis was performed using a FaCScan (BectonDickinson), and the data were analyzed using the Cell Quest program.

D) Expression of P703P in Baculovirus

The cDNA for full-length P703P-DE5 (SEQ ID NO: 326), together withseveral flanking restriction sites, was obtained by digesting theplasmid pCDNA703 with restriction endonucleases Xba I and Hind III. Theresulting restriction fragment (approx. 800 base pairs) was ligated intothe transfer plasmid pFastBacl which was digested with the samerestriction enzymes. The sequence of the insert was confirmed by DNAsequencing. The recombinant transfer plasmid pFBP703 was used to makerecombinant bacmid DNA and baculovirus using the Bac-To-Bac Baculovirusexpression system (BRL Life Technologies). High Five cells were infectedwith the recombinant virus BVP703, as described above, to obtainrecombinant P703P protein.

E) Expression of P788P in E. coli

A truncated, N-terminal portion, of P788P (residues 1-644 of SEQ ID NO:777; referred to as P788P-N) fused with a C-terminal 6×His Tag wasexpressed in E. coli as follows. P788P cDNA was amplified using theprimers AW080 and AW081 (SEQ ID NO: 815 and 816). AW080 is a sensecloning primer with an NdeI site. AW081 is an antisense cloning primerwith a XhoI site. The PCR-amplified P788P, as well as the vector pCRX1,were digested with NdeI and XhoI. Vector and insert were ligated andtransformed into NovaBlue cells. Colonies were randomly screened forinsert and then sequenced. P788P-N clone #6 was confirmed to beidentical to the designed construct. The expression construct P788P-N#6/pCRX1 was transformed into E. coli BL21 CodonPlus-RIL competentcells. After induction, most of the cells grew well, achieving OD600 ofgreater than 2.0 after 3 hr. Coomassie stained SDS-PAGE showed anover-expressed band at about 75 kD. Western blot analysis using a6×HisTag antibody confirmed the band was P788P-N. The determined cDNAsequence for P788P-N is provided in SEQ ID NO: 817, with thecorresponding amino acid sequence being provided in SEQ ID NO: 818.

F) Expression of P510S in E. coli

The p510S protein has 9 potential transmembrane domains and is predictedto be located at the plasma membrane. The C-terminal protein of thisprotein, as well as the predicted third extracellular domain of p510Swere expressed in E. coli as follows.

The expression construct referred to as Ra12-P501S-C was designed tohave a 6 HisTag at the N-terminal enc, followed by the M. tuberculosisantigen Ra12 (SEQ ID NO: 819) and then the C-terminal portion of P510S(amino residues 1176-1261 of SEQ ID NO: 538). Full-length P510S was usedto amplify the P510S-C fragment by PCR using the primers AW056 and AW057(SEQ ID NO: 820 and 821, respectively). AW056 is a sense cloning primerwith an EcoRI site. AW057 is an antisense primer with stop and XhoIsites. The amplified P501S fragment and Ra12/pCRX1 were digested withEcoRI and XhoI and then purified. The insert and vector were ligatedtogether and transformed into NovaBlue. Colonies were randomly screenedfor insert and sequences. For protein expression, the expressionconstruct was transformed into E. coli BL21 (DE3) CodonPlus-RILcompetent cells. A mini-induction screen was performed to optimize theexpression conditions. After induction the cells grew well, achieving OD600 nm greater than 2.0 after 3 hours. Coomassie stain SDS-PAGE showed ahighly over-expressed band at approx. 30 kD. Though this is higher thanthe expected molecular weight, western blot analysis was positive,showing this band to be the His tag-containing protein. The optimizedculture conditions are as follows. Dilute overnight culture/daytimeculture (LB+kanamycin+hloramphenicol) into 2×YT (with kanamycin andchloramphenicol) at a ratio of 25 ml ulture to 1 liter 2×YT. Allow togrow at 37° C. until OD600=0.6. Take an aliquot out as T0 sample. Add 1mM IPTG and allow to grow at 30° C. for 3 hours. Take out a T3 sample,spin down cells and store at −80° C. The determined cDNA and amino acidsequences for the Ra12-P510S-C construct are provided in SEQ ID NO: 822and 825, respectively.

The expression construct p510S-C was designed to have a 5′ added startcodon and a glycine (GGA) codon and then the P510S C terminal fragmentfollowed by the in frame 6×histidine tag and stop codon from the pET28bvector. The cloning strategy is similar to that used for Ra12-P510S-C,except that the PCR primers employed were those shown in SEQ ID NO: 828and 829, respectively and the NcoI/XhoI cut in pET28b was used. Theprimer of SEQ ID NO: 828 created a 5′ NcoI site and added a start codon.The antisense primer of SEQ ID NO: 829 creates a XhoI site on P510S Cterminal fragment. Clones were confirmed by sequencing. For proteinexpression, the expression construct was transformed into E. coli BL21(DE3) CodonPlus-RIL competent cells. An OD600 of greater than 2.0 wasobtained 30 hours after induction. Coomassie stained SDS-PAGE showed anover-expressed band at about 11 kD. Western blot analysis confirmed thatthe band was P510S-C, as did N-terminal protein sequencing. Theoptimized culture conditions are as follows: dilute overnightculture/daytime culture (LB+kanamycin+chloramphenicol) into 2×YT(+kanamycin and chloramphenicol) at a ratio of 25 mL culture to 1 liter2×YT, and allow to grow at 37° C. until an OD 600 of about 0.5 isreached. Take out an aliquot as T0 sample. Add 1 mM IPTG and allow togrow at 30° C. for 3 hours. Spin down the cells and store at −80° C.until purification. The determined cDNA and amino acid sequence for theP510S-C construct are shown in SEQ ID NO: 823 and 826, respectively.

The predicted third extracellular domain of p510S (p510S-E3; residues328-676 of SEQ ID NO: 538) was expressed in E. coli as follows. TheP510S fragment was amplified by PCR using the primers shown in SEQ IDNO: 830 and 831. The primer of SEQ ID NO: 830 is a sense primer with anNdeI site for use in ligating into pPDM. The primer of SEQ ID NO: 831 isan antisense primer with an added XhoI site for use in ligating intopPDM. The resulting fragment was cloned to pPDM at the NdeI and XhoIsites. Clones were confirmed by sequencing. For protein expression, theclone ws transformed into E. coli BL21 (DE3) CodonPlus-RIL competentcells. After induction, an OD600 of greater than 2.0 was achieved after3 hours. Coomassie stained SDS-PAGE showed an over-expressed band atabout 39 kD, and N-terminal sequencing confirmed the N-terminal to bethat of p510S-E3. Optimized culture conditions are as follows: diluteovernight culture/daytime culture (LB+kanamycin+chloramphenicol) into2×YT (kanamycin and chloramphenicol) at a ratio of 25 ml culture to 1liter 2×YT. Allow to grow at 37° C. until OD 600 equals 0.6. Take out analiquot as T0 sample. Add 1 mM IPTG and allow to grow at 30° C. for 3hours. Take out a T3 sample, spin down the cells and store at −80° C.until purification. The determined cDNA and amino acid sequences for theP501S-E3 construct are provided in SEQ ID NO: 824 and 827, respectively.

G) Expression of P775S in E. coli

The antigen P775P contains multiple open reading frames (ORF). The thirdORF, encoding the protein of SEQ ID NO: 483, has the best emotif score.An expression fusion construct containing the M. tuberculosis antigenRa12 (SEQ ID NO: 819) and P775P-ORF3 with an N-terminal 6×HisTag wasprepared as follows. P775P-ORF3 was amplified using the sense PCRprimers of SEQ ID NO: 832 and the anti-sense PCR primer of SEQ ID NO:833. The PCR amplified fragment of P775P and Ra12/pCRX1 were digestedwith the restriction enzymes EcoRI and XhoI. Vector and insert wereligated and then transformed into NovaBlue cells. Colonies were randomlyscreened for insert and then sequenced. A clone having the desiredsequence was transformed into E. coli BL21 (DE3) CodonPlus-RIL competentcells. Two hours after induction, the cell density peaked at OD600 ofapproximately 1.8. Coomassie stained SDS-PAGE showed an over-expressedband at about 31 kD. Western blot using 6×HisTag antibody confirmed thatthe band was Ra12-P775P-ORF3. The determined cDNA and amino acidsequences for the fusion construct are provided in SEQ ID NO: 834 and835, respectively.

H) Expression of a P703P His tag Fusion Protein in E. coli

The cDNA for the coding region of P703P was prepared by PCR using theprimers of SEQ ID NO: 836 and 837. The PCR product was digested withEcoRI restriction enzyme, gel purified and cloned into a modified pET28vector with a His tag in frame, which had been digested with Eco72I andEcoRI restriction enzymes. The correct construct was confirmed by DNAsequence analysis and then transformed into E. coli BL21 (DE3) pLys Sexpression host cells. The determined amino acid and cDNA sequences forthe expressed recombinant P703P are provided in SEQ ID NO: 838 and 839,respectively.

I) Expression of a P705P His tag Fusion Protein in E. coli

The cDNA for the coding region of P705P was prepared by PCR using theprimers of SEQ ID NO: 840 and 841. The PCR product was digested withEcoRI restriction enzyme, gel purified and cloned into a modified pET28vector with a His tag in frame, which had been digested with Eco72I andEcoRI restriction enzymes. The correct construct was confirmed by DNAsequence analysis and then transformed into E. coli BL21 (DE3) pLys Sand BL21 (DE3) CodonPlus expression host cells. The determined aminoacid and cDNA sequences for the expressed recombinant P705P are providedin SEQ ID NO: 842 and 843, respectively.

J) Expression of a P711P His tag Fusion Protein in E. coli

The cDNA for the coding region of P711P was prepared by PCR using theprimers of SEQ ID NO: 844 and 845. The PCR product was digested withEcoRI restriction enzyme, gel purified and cloned into a modified pET28vector with a His tag in frame, which had been digested with Eco72 I andEcoRI restriction enzymes. The correct construct was confirmed by DNAsequence analysis and then transformed into E. Coli BL21 (DE3) pLys Sand BL21 (DE3) CodonPlus expression host cells. The determined aminoacid and cDNA sequences for the expressed recombinant P711 P areprovided in SEQ ID NO: 846 and 847, respectively.

EXAMPLE 18 Preparation and Characterization of Antibodies AgainstProstate-specific Polypeptides

A) Preparation and Characterization of Polyclonal Antibodies AgainstP703P. P504S and P509S

Polyclonal antibodies against P703P, P504S and P509S were prepared asfollows.

Each prostate tumor antigen expressed in an E. coli recombinantexpression system was grown overnight in LB broth with the appropriateantibiotics at 37° C. in a shaking incubator. The next morning, 10 ml ofthe overnight culture was added to 500 ml to 2×YT plus appropriateantibiotics in a 2L-baffled Erlenmeyer flask. When the Optical Density(at 560 nm) of the culture reached 0.4-0.6, the cells were induced withIPTG (1 mM). Four hours after induction with IPTG, the cells wereharvested by centrifugation. The cells were then washed with phosphatebuffered saline and centrifuged again. The supernatant was discarded andthe cells were either frozen for future use or immediately processed.Twenty ml of lysis buffer was added to the cell pellets and vortexed. Tobreak open the E. coli cells, this mixture was then run through theFrench Press at a pressure of 16,000 psi. The cells were thencentrifuged again and the supernatant and pellet were checked bySDS-PAGE for the partitioning of the recombinant protein. For proteinsthat localized to the cell pellet, the pellet was resuspended in 10 mMTris pH 8.0, 1% CHAPS and the inclusion body pellet was washed andcentrifuged again. This procedure was repeated twice more. The washedinclusion body pellet was solubilized with either 8 M urea or 6 Mguanidine HCl containing 10 mM Tris pH 8.0 plus 10 mM imidazole. Thesolubilized protein was added to 5 ml of nickel-chelate resin (Qiagen)and incubated for 45 min to 1 hour at room temperature with continuousagitation. After incubation, the resin and protein mixture were pouredthrough a disposable column and the flow through was collected. Thecolumn was then washed with 10-20 column volumes of the solubilizationbuffer. The antigen was then eluted from the column using 8M urea, 10 mMTris pH 8.0 and 300 mM imidazole and collected in 3 ml fractions. ASDS-PAGE gel was run to determine which fractions to pool for furtherpurification.

As a final purification step, a strong anion exchange resin such asHiPrepQ (Biorad) was equilibrated with the appropriate buffer and thepooled fractions from above were loaded onto the column. Each antigenwas eluted off the column with a increasing salt gradient. Fractionswere collected as the column was run and another SDS-PAGE gel was run todetermine which fractions from the column to pool. The pooled fractionswere dialyzed against 10 mM Tris pH 8.0. The proteins were then vialedafter filtration through a 0.22 micron filter and the antigens werefrozen until needed for immunization.

Four hundred micrograms of each prostate antigen was combined with 100micrograms of muramyldipeptide (MDP). Every four weeks rabbits wereboosted with 100 micrograms mixed with an equal volume of IncompleteFreund's Adjuvant (IFA). Seven days following each boost, the animal wasbled. Sera was generated by incubating the blood at 4° C. for 12-4 hoursfollowed by centrifugation.

Ninety-six well plates were coated with antigen by incubating with 50microliters (typically 1 microgram) of recombinant protein at 4° C. for20 hours. 250 microliters of BSA blocking buffer was added to the wellsand incubated at room temperature for 2 hours. Plates were washed 6times with PBS/0.01% Tween. Rabbit sera was diluted in PBS. Fiftlymicroliters of diluted sera was added to each well and incubated at roomtemperature for 30 min. Plates were washed as described above before 50microliters of goat anti-rabbit horse radish peroxidase (HRP) at a1:10000 dilution was added and incubated at room temperature for 30 min.Plates were again washed as described above and 100 microliters of TMBmicrowell peroxidase substrate was added to each well. Following a 15min incubation in the dark at room temperature, the colorimetricreaction was stopped with 100 microliters of 1N H₂SO₄ and readimmediately at 450 nm. All polyclonal antibodies showed immunoreactivityto the appropriate antigen.

B) Preparation and Characterization of Antibodies Against P501S

A murine monoclonal antibody directed against the carboxy-terminus ofthe prostate-specific antigen P501S was prepared as follows.

A truncated fragment of P501S (amino acids 355-526 of SEQ ID NO: 113)was generated and cloned into the pET28b vector (Novagen) and expressedin E. coli as a thioredoxin fusion protein with a histidine tag. Thetrx-P501S fusion protein was purified by nickel chromatography, digestedwith thrombin to remove the trx fragment and further purified by an acidprecipitation procedure followed by reverse phase HPLC.

Mice were immunized with truncated P501S protein. Serum bleeds from micethat potentially contained anti-P501S polyclonal sera were tested forP501S-specific reactivity using ELISA assays with purified P501S andtrx-P501S proteins. Serum bleeds that appeared to react specificallywith P501S were then screened for P501S reactivity by Western analysis.Mice that contained a P501S-specific antibody component were sacrificedand spleen cells were used to generate anti-P501S antibody producinghybridomas using standard techniques. Hybridoma supernatants were testedfor P501S-specific reactivity initially by ELISA, and subsequently byFACS analysis of reactivity with P501S transduced cells. Based on theseresults, a monoclonal hybridoma referred to as 10E3 was chosen forfurther subcloning. A number of subclones were generated, tested forspecific reactivity to P501S using ELISA and typed for IgG isotype. Theresults of this analysis are shown below in Table V. Of the 16 subclonestested, the monoclonal antibody 10E3-G4-D3 was selected for furtherstudy.

TABLE V Isotype analysis of murine anti-P501S monoclonal antibodiesHybridoma clone Isotype Estimated [Ig] in supernatant (μg/ml) 4D11 IgG114.6 1G1 IgG1 0.6 4F6 IgG1 72 4H5 IgG1 13.8 4H5-E12 IgG1 10.7 4H5-EH2IgG1 9.2 4H5-H2-A10 IgG1 10 4H5-H2-A3 IgG1 12.8 4H5-H2-A10-G6 IgG1 13.64H5-H2-B11 IgG1 12.3 10E3 IgG2a 3.4 10E3-D4 IgG2a 3.8 10E3-D4-G3 IgG2a9.5 10E3-D4-G6 IgG2a 10.4 10E3-E7 IgG2a 6.5 8H12 IgG2a 0.6

The specificity of 10E3-G4-D3 for P501S was examined by FACS analysis.Specifically, cells were fixed (2% formaldehyde, 10 minutes),permeabilized (0.1% saponin, 10 minutes) and stained with 10E3-G4-D3 at0.5-1 μg/ml, followed by incubation with a secondary, FITC-conjugatedgoat anti-mouse Ig antibody (Pharmingen, San Diego, Calif.). Cells werethen analyzed for FITC fluorescence using an Excalibur fluorescenceactivated cell sorter. For FACS analysis of transduced cells, B-LCL wereretrovirally transduced with P501S. For analysis of infected cells,B-LCL were infected with a vaccinia vector that expresses P501S. Todemonstrate specificity in these assays, B-LCL transduced with adifferent antigen (P703P) and uninfected B-LCL vectors were utilized.10E3-G4-D3 was shown to bind with P501S-transduced B-LCL and also withP501S-infected B-LCL, but not with either uninfected cells orP703P-transduced cells.

To determine whether the epitope recognized by 10E3-G4-D3 was found onthe surface or in an intracellular compartment of cells, B-LCL weretransduced with P501S or HLA-B8 as a control antigen and either fixedand permeabilized as described above or directly stained with 10E3-G4-D3and analyzed as above. Specific recognition of P501S by 10E3-G4-D3 wasfound to require permneabilization, suggesting that the epitoperecognized by this antibody is intracellular.

The reactivity of 10E3-G4-D3 with the three prostate tumor cell linesLncap, PC-3 and DU-145, which are known to express high, medium and verylow levels of P501S, respectively, was examined by permeabilizing thecells and treating them as described above. Higher reactivity of10E3-G4-D3 was seen with Lncap than with PC-3, which in turn showedhigher reactivity that DU-145. These results are in agreement with thereal time PCR and demonstrate that the antibody specifically recognizesP501S in these tumor cell lines and that the epitope recognized inprostate tumor cell lines is also intracellular.

Specificity of 10E3-G4-D3 for P501S was also demonstrated by Westernblot analysis. Lysates from the prostate tumor cell lines Lncap, DU-145and PC-3, from P501S-transiently transfected HEK293 cells, and fromnon-transfected HEK293 cells were generated. Western blot analysis ofthese lysates with 10E3-G4-D3 revealed a 46 kDa immunoreactive band inLncap, PC-3 and P501S-transfected HEK cells, but not in DU-145 cells ornon-transfected HEK293 cells. P501S mRNA expression is consistent withthese results since semi-quantitative PCR analysis revealed that P501SmRNA is expressed in Lncap, to a lesser but detectable level in PC-3 andnot at all in DU-145 cells. Bacterially expressed and purifiedrecombinant P501S (referred to as P501SStr2) was recognized by10E3-G4-D3 (24 kDa), as was full-length P501S that was transientlyexpressed in HEK293 cells using either the expression vector VR1012 orpCEP4. Although the predicted molecular weight of P501S is 60.5 kDa,both transfected and “native” P501S run at a slightly lower mobility dueto its hydrophobic nature.

Immunohistochemical analysis was performed on prostate tumor and a panelof normal tissue sections (prostate, adrenal, breast, cervix, colon,duodenum, gall bladder, ileum, kidney, ovary, pancreas, parotid gland,skeletal muscle, spleen and testis). Tissue samples were fixed informalin solution for 24 hours and embedded in paraffin before beingsliced into 10 micron sections. Tissue sections were permeabilized andincubated with 10E3-G4-D3 antibody for 1 hr. HRP-labeled anti-mousefollowed by incubation with DAB chromogen was used to visualize P501Simmunoreactivity. P510S was found to be highly expressed in both normalprostate and prostate tumor tissue but was not detected in any of theother tissues tested.

To identify the epitope recognized by 10E3-G4-D3, an epitope mappingapproach was pursued. A series of 13 overlapping 20-21 mers (5 aminoacid overlap; SEQ ID NO: 489-501) was synthesized that spanned thefragment of P501S used to generate 10E3-G4-D3. Flat bottom 96 wellmicrotiter plates were coated with either the peptides or the P501Sfragment used to immunize mice, at 1 microgram/ml for 2 hours at 37° C.Wells were then aspirated and blocked with phosphate buffered salinecontaining 1% (w/v) BSA for 2 hours at room temperature, andsubsequently washed in PBS containing 0.1% Tween 20 (PBST). Purifiedantibody 10E3-G4-D3 was added at 2 fold dilutions (1000 ng-16 ng) inPBST and incubated for 30 minutes at room temperature. This was followedby washing 6 times with PBST and subsequently incubating withHRP-conjugated donkey anti-mouse IgG (H+L)Affinipure F(ab′) fragment(Jackson Immunoresearch, West Grove, Pa.) at 1:20000 for 30 minutes.Plates were then washed and incubated for 15 minutes in tetramethylbenzidine. Reactions were stopped by the addition of 1N sulfuric acidand plates were read at 450 nm using an ELISA plate reader. As shown inFIG. 8, reactivity was seen with the peptide of SEQ ID NO: 496(corresponding to amino acids 439-459 of P510S) and with the P501Sfragment but not with the remaining peptides, demonstrating that theepitope recognized by 10E3-G4-D3 is localized to amino acids 439-459 ofSEQ ID NO: 113.

In order to further evaluate the tissue specificity of P501S,multi-array immunohistochemical analysis was performed on approximately4700 different human tissues encompassing all the major normal organs aswell as neoplasias derived from these tissues. Sixty-five of these humantissue samples were of prostate origin. Tissue sections 0.6 mm indiameter were formalin-fixed and paraffin embedded. Samples werepretreated with HIER using 10 mM citrate buffer pH 6.0 and boiling for10 min. Sections were stained with 10E3-G4-D3 and P501S immunoreactivitywas visualized with HRP. All the 65 prostate tissues samples (5 normal,55 untreated prostate tumors, 5 hormone refractory prostate tumors) werepositive, showing distinct perinuclear staining. All other tissuesexamined were negative for P501S expression.

C) Preparation and Characterization of Antibodies Against P503S

A fragment of P503S (amino acids 113-241 of SEQ ID NO: 114) wasexpressed and purified from bacteria essentially as described above forP501S and used to immunize both rabbits and mice. Mouse monoclonalantibodies were isolated using standard hybridoma technology asdescribed above. Rabbit monoclonal antibodies were isolated usingSelected Lymphocyte Antibody Method (SLAM) technology at ImmgenicsPharmaceuticals (Vancouver, BC, Canada). Table VI, below, lists themonoclonal antibodies that were developed against P503S.

TABLE VI Antibody Species 20D4 Rabbit JA1 Rabbit 1A4 Mouse 1C3 Mouse 1C9Mouse 1D12 Mouse 2A11 Mouse 2H9 Mouse 4H7 Mouse 8A8 Mouse 8D10 Mouse9C12 Mouse 6D12 Mouse

The DNA sequences encoding the complementarity determining regions(CDRs) for the rabbit monoclonal antibodies 20D4 and JA1 were determinedand are provided in SEQ ID NO: 502 and 503, respectively.

In order to better define the epitope binding region of each of theantibodies, a series of overlapping peptides were generated that spanamino acids 109-213 of SEQ ID NO: 114. These peptides were used toepitope map the anti-P503S monoclonal antibodies by ELISA as follows.The recombinant fragment of P503S that was employed as the immunogen wasused as a positive control. Ninety-six well microtiter plates werecoated with either peptide or recombinant antigen at 20 ng/wellovernight at 4° C. Plates were aspirated and blocked with phosphatebuffered saline containing 1% (w/v) BSA for 2 hours at room temperaturethen washed in PBS containing 0.1% Tween 20 (PBST). Purified rabbitmonoclonal antibodies diluted in PBST were added to the wells andincubated for 30 min at room temperature. This was followed by washing 6times with PBST and incubation with Protein-A HRP conjugate at a 1:2000dilution for a further 30 min. Plates were washed six times in PBST andincubated with tetramethylbenzidine (TMB) substrate for a further 15min. The reaction was stopped by the addition of IN sulfuric acid andplates were read at 450 nm using at ELISA plate reader. ELISA with themouse monoclonal antibodies was performed with supernatants from tissueculture run neat in the assay.

All of the antibodies bound to the recombinant P503S fragment, with theexception of the negative control SP2 supernatant. 20D4, JA1 and 1D12bound strictly to peptide #2101 (SEQ ID NO: 504), which corresponds toamino acids 151-169 of SEQ ID NO: 114. 1C3 bound to peptide #2102 (SEQID NO: 505), which corresponds to amino acids 165-184 of SEQ ID NO: 114.9C12 bound to peptide #2099 (SEQ ID NO: 522), which corresponds to aminoacids 120-139 of SEQ ID NO: 114. The other antibodies bind to regionsthat were not examined in these studies.

Subsequent to epitope mapping, the antibodies were tested by FACSanalysis on a cell line that stably expressed P503S to confirm that theantibodies bind to cell surface epitopes. Cells stably transfected witha control plasmid were employed as a negative control. Cells werestained live with no fixative. 0.5 μg of anti-P503S monoclonal antibodywas added and cells were incubated on ice for 30 min before being washedtwice and incubated with a FITC-labelled goat anti-rabbit or mousesecondary antibody for 20 min. After being washed twice, cells wereanalyzed with an Excalibur fluorescent activated cell sorter. Themonoclonal antibodies 1C3, 1D12, 9C12, 20D4 and JA1, but not 8D3, werefound to bind to a cell surface epitope of P503S.

In order to determine which tissues express P503S, immunohistochemicalanalysis was performed, essentially as described above, on a panel ofnormal tissues (prostate, adrenal, breast, cervix, colon, duodenum, gallbladder, ileum, kidney, ovary, pancreas, parotid gland, skeletal muscle,spleen and testis). HRP-labeled anti-mouse or anti-rabbit antibodyfollowed by incubation with TMB was used to visualize P503Simmunoreactivity. P503S was found to be highly expressed in prostatetissue, with lower levels of expression being observed in cervix, colon,ileum and kidney, and no expression being observed in adrenal, breast,duodenum, gall bladder, ovary, pancreas, parotid gland, skeletal muscle,spleen and testis.

Western blot analysis was used to characterize anti-P503S monoclonalantibody specificity. SDS-PAGE was performed on recombinant (rec) P503Sexpressed in and purified from bacteria and on lysates from HEK293 cellstransfected with full length P503S. Protein was transferred tonitrocellulose and then Western blotted with each of the anti-P503Smonoclonal antibodies (20D4, JA1, 1D12, 6D12 and 9C12) at an antibodyconcentration of 1 μg/ml. Protein was detected using horse radishperoxidase (HRP) conjugated to either a goat anti-mouse monoclonalantibody or to protein A-sepharose. The monoclonal antibody 20D4detected the appropriate molecular weight 14 kDa recombinant P503S(amino acids 113-241) and the 23.5 kDa species in the HEK293 celllysates transfected with full length P503S. Other anti-P503S monoclonalantibodies displayed similar specificity by Western blot.

D) Preparation and Characterization of Antibodies Against P703P

Rabbits were immunized with either a truncated (P703Ptr1; SEQ ID NO:172) or full-length mature form (P703Pf1; SEQ ID NO: 523) of recombinantP703P protein was expressed in and purified from bacteria as describedabove. Affinity purified polyclonal antibody was generated usingimmunogen P703Pf1 or P703Ptr1 attached to a solid support. Rabbitmonoclonal antibodies were isolated using SLAM technology at mmgenicsPharmaceuticals. Table VII below lists both the polyclonal andmonoclonal ntibodies that were generated against P703P.

TABLE VII Antibody Immunogen Species/type Aff. Purif. P703P (truncated);#2594 P703Ptr1 Rabbit polyclonal Aff. Purif. P703P (full length); #9245P703Pf1 Rabbit polyclonal 2D4 P703Ptr1 Rabbit monoclonal 8H2 P703Ptr1Rabbit monoclonal 7H8 P703Ptr1 Rabbit monoclonal

The DNA sequences encoding the complementarity determining regions(CDRs) for the rabbit monoclonal antibodies 8H2, 7H8 and 2D4 weredetermined and are provided in SEQ ID NO: 506-508, respectively.

Epitope mapping studies were performed as described above. Monoclonalantibodies 2D4 and 7H8 were found to specifically bind to the peptidesof SEQ ID NO: 509 (corresponding to amino acids 145-159 of SEQ ID NO:172) and SEQ ID NO: 510 (corresponding to amino acids 11-25 of SEQ IDNO: 172), respectively. The polyclonal antibody 2594 was found to bindto the peptides of SEQ ID NO: 511-514, with the polyclonal antibody 9427binding to the peptides of SEQ ID NO: 515-517.

The specificity of the anti-P703P antibodies was determined by Westernblot analysis as follows. SDS-PAGE was performed on (1) bacteriallyexpressed recombinant antigen; (2) lysates of HEK293 cells and Ltk-/−cells either untransfected or transfected with a plasmid expressing fulllength P703P; and (3) supernatant isolated from these cell cultures.Protein was transferred to nitrocellulose and then Western blotted usingthe anti-P703P polyclonal antibody #2594 at an antibody concentration of1 μg/ml. Protein was detected using horse radish peroxidase (HRP)conjugated to an anti-rabbit antibody. A 35 kDa immunoreactive bandcould be observed with recombinant P703P. Recombinant P703P runs at aslightly higher molecular weight since it is epitope tagged. In lysatesand supernatants from cells transfected with full length P703P, a 30 kDaband corresponding to P703P was observed. To assure specificity, lysatesfrom HEK293 cells stably transfected with a control plasmid were alsotested and were negative for P703P expression. Other anti-P703Pantibodies showed similar results.

Immunohistochemical studies were performed as described above, usinganti-P703P monoclonal antibody. P703P was found to be expressed at highlevels in normal prostate and prostate tumor tissue but was notdetectable in all other tissues tested (breast tumor, lung tumor andnormal kidney).

EXAMPLE 19 Characterization of Cell Surface Expression and ChromosomeLocalization of the Prostate-specific AntigeniP501S

This example describes studies demonstrating that the prostate-specificantigen P501S is expressed on the surface of cells, together withstudies to determine the probable chromosomal location of P501S.

The protein P501S (SEQ ID NO: 113) is predicted to have 11 transmembranedomains. Based on the discovery that the epitope recognized by theanti-P501S monoclonal antibody 10E3-G4-D3 (described above in Example17) is intracellular, it was predicted that following transmembranedeterminants would allow the prediction of extracellular domains ofP501S. FIG. 9 is a schematic representation of the P501S protein showingthe predicted location of the transmembrane domains and theintracellular epitope described in Example 17. Underlined sequencerepresents the predicted transmembrane domains, bold sequence representsthe predicted extracellular domains, and italicized sequence representsthe predicted intracellular domains. Sequence that is both bold andunderlined represents sequence employed to generate polyclonal rabbitserum. The location of the transmembrane domains was predicted usingHHMTOP as described by Tusnady and Simon (Principles Governing AminoAcid Composition of Integral Membrane Proteins: Applications to TopologyPrediction, J. Mol. Biol. 283:489-506, 1998).

Based on FIG. 9, the P501S domain flanked by the transmembrane domainscorresponding to amino acids 274-295 and 323-342 is predicted to beextracellular. The peptide of SEQ ID NO: 518 corresponds to amino acids306-320 of P501S and lies in the predicted extracellular domain. Thepeptide of SEQ ID NO: 519, which is identical to the peptide of SEQ IDNO: 518 with the exception of the substitution of the histidine with anasparginine, was synthesized as described above. A Cys-Gly was added tothe C-termninus of the peptide to facilitate conjugation to the carrierprotein. Cleavage of the peptide from the solid support was carried outusing the following cleavage mixture: trifluoroaceticacid:ethanediol:thioanisol:water:phenol (40:1:2:2:3). After cleaving fortwo hours, the peptide was precipitated in cold ether. The peptidepellet was then dissolved in 10% v/v acetic acid and lyophilized priorto purification by C18 reverse phase hplc. A gradient of 5-60%acetonitrile (containing 0.05% TFA) in water (containing 0.05% TFA) wasused to elute the peptide. The purity of the peptide was verified byhplc and mass spectrometry, and was determined to be >95%. The purifiedpeptide was used to generate rabbit polyclonal antisera as describedabove.

Surface expression of P501S was examined by FACS analysis. Cells werestained with the polyclonal anti-P510S peptide serum at 10 μg/ml,washed, incubated with a secondary FITC-conjugated goat anti-rabbit Igantibody (ICN), washed and analyzed for FITC fluorescence using anExcalibur fluorescence activated cell sorter. For FACS analysis oftransduced cells, B-LCL were retrovirally transduced with P501S. Todemonstrate specificity in these assays, B-LCL transduced with anirrelevant antigen (P703P) or nontransduced were stained in parallel.For FACS analysis of prostate tumor cell lines, Lncap, PC-3 and DU-145were utilized. Prostate tumor cell lines were dissociated from tissueculture plates using cell dissociation medium and stained as above. Allsamples were treated with propidium iodide (PI) prior to FACS analysis,and data was obtained from PI-excluding (i.e., intact andnon-permeabilized) cells. The rabbit polyclonal serum generated againstthe peptide of SEQ ID NO: 519 was shown to specifically recognize thesurface of cells transduced to express P501S, demonstrating that theepitope recognized by the polyclonal serum is extracellular.

To determine biochemically if P501S is expressed on the cell surface,peripheral membranes from Lncap cells were isolated and subjected toWestern blot analysis. Specifically, Lncap cells were lysed using adounce homogenizer in 5 ml of homogenization buffer (250 mM sucrose, 10mM HEPES, 1 mM EDTA, pH 8.0, 1 complete protease inhibitor tablet(Boehringer Mannheim)). Lysate samples were spun at 1000 g for 5 min at4° C. The supernatant was then spun at 8000 g for 10 min at 4° C.Supernatant from the 8000 g spin was recovered and subjected to a100,000 g spin for 30 min at 4° C. to recover peripheral membrane.Samples were then separated by SDS-PAGE and Western blotted with themouse monoclonal antibody 10E3-G4-D3 (described above in Example 17)using conditions described above. Recombinant purified P501S, as well asHEK293 cells transfected with and over-expressing P501S were included aspositive controls for P501S detection. LCL cell lysate was included as anegative control. P501S could be detected in Lncap total cell lysate,the 8000 g (internal membrane) fraction and also in the 100,000 g(plasma membrane) fraction. These results indicate that P501S isexpressed at, and localizes to, the peripheral membrane.

To demonstrate that the rabbit polyclonal antiserum generated to thepeptide of SEQ ID NO: 519 specifically recognizes this peptide as wellas the corresponding native peptide of SEQ ID NO: 518, ELISA analyseswere performed. For these analyses, flat-bottomed 96 well microtiterplates were coated with either the peptide of SEQ ID NO: 519, the longerpeptide of SEQ ID NO: 520 that spans the entire predicted extracellulardomain, the peptide of SEQ ID NO: 521 which represents the epitoperecognized by the P501S-specific antibody 10E3-G4-D3, or a P501Sfragment (corresponding to amino acids 355-526 of SEQ ID NO: 113) thatdoes not include the immunizing peptide sequence, at 1 μg/ml for 2 hoursat 37° C. Wells were aspirated, blocked with phosphate buffered salinecontaining 1% (w/v) BSA for 2 hours at room temperature and subsequentlywashed in PBS containing 0.1% Tween 20 (PBST). Purified anti-P501Spolyclonal rabbit serum was added at 2 fold dilutions (1000 ng-125 ng)in PBST and incubated for 30 min at room temperature. This was followedby washing 6 times with PBST and incubating with HRP-conjugated goatanti-rabbit IgG (H+L) Affinipure F(ab′) fragment at 1:20000 for 30 min.Plates were then washed and incubated for 15 min in tetramethylbenzidine. Reactions were stopped by the addition of 1N sulfuric acidand plates were read at 450 nm using an ELISA plate reader. As shown inFIG. 11, the anti-P501S polyclonal rabbit serum specifically recognizedthe peptide of SEQ ID NO: 519 used in the immunization as well as thelonger peptide of SEQ ID NO: 520, but did not recognize the irrelevantP501S-derived peptides and fragments.

In further studies, rabbits were immunized with peptides derived fromthe P501S sequence and predicted to be either extracellular orintracellular, as shown in FIG. 9. Polyclonal rabbit sera were isolatedand polyclonal antibodies in the serum were purified, as describedabove. To determine specific reactivity with P501S, FACS analysis wasemployed, utilizing either B-LCL transduced with P501S or the irrelevantantigen P703P, of B-LCL infected with vaccinia virus-expressing P501S.For surface expression, dead and non-intact cells were excluded from theanalysis as described above. For intracellular staining, cells werefixed and permeabilized as described above. Rabbit polyclonal serumgenerated against the peptide of SEQ ID NO: 548, which corresponds toamino acids 181-198 of P501S, was found to recognize a surface epitopeof P501S. Rabbit polyclonal serum generated against the peptide SEQ IDNO: 551, which corresponds to amino acids 543-553 of P501S, was found torecognize an epitope that was either potentially extracellular orintracellular since in different experiments intact or permeabilizedcells were recognized by the polyclonal sera. Based on similar deductivereasoning, the sequences of SEQ ID NO: 541-547, 549 and 550, whichcorrespond to amino acids 109-122, 539-553, 509-520, 37-54, 342-359,295-323, 217-274, 143-160 and 75-88, respectively, of P501S, can beconsidered to be potential surface epitopes of P501S recognized byantibodies.

The chromosomal location of P501S was determined using the GeneBridge 4Radiation Hybrid panel (Research Genetics). The PCR primers of SEQ IDNO: 528 and 529 were employed in PCR with DNA pools from the hybridpanel according to the manufacturer's directions. After 38 cycles ofamplification, the reaction products were separated on a 1.2% agarosegel, and the results were analyzed through the Whitehead Institute/MITCenter for Genome Research web server(http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.p1) to determinethe probable chromosomal location. Using this approach, P501S was mappedto the long arm of chromosome 1 at WI-9641 between q32 and q42. Thisregion of chromosome 1 has been linked to prostate cancer susceptibilityin hereditary prostate cancer (Smith et al. Science 274:1371-1374, 1996and Berthon et al. Am. J. Hum. Genet. 62:1416-1424, 1998). These resultssuggest that P501S may play a role in prostate cancer malignancy.

EXAMPLE 20 Regulation of Expression of the Prostate-specific AntigenP501S

Steroid (androgen) hormone modulation is a common treatment modality inprostate cancer. The expression of a number of prostate tissue-specificantigens have previously been demonstrated to respond to androgen. Theresponsiveness of the prostate-specific antigen P501S to androgentreatment was examined in a tissue culture system as follows.

Cells from the prostate tumor cell line LNCaP were plated at 1.5×10⁶cells/T75 flask (for RNA isolation) or 3×10⁵ cells/well of a 6-wellplate (for FACS analysis) and grown overnight in RPMI 1640 mediacontaining 10% charcoal-stripped fetal calf serum (BRL LifeTechnologies, Gaithersburg, Md.). Cell culture was continued for anadditional 72 hours in RPMI 1640 media containing 10% charcoal-strippedfetal calf serum, with 1 nM of the synthetic androgen Methyltrienolone(R1881; New England Nuclear) added at various time points. Cells werethen harvested for RNA isolation and FACS analysis at 0, 1, 2, 4, 8, 16,24, 28 and 72-hours post androgen addition. FACS analysis was performedusing the anti-P510S antibody 10E3-G4-D3 and permeabilized cells.

For Northern analysis, 5-10 micrograms of total RNA was run on aformaldehyde denaturing gel, transferred to Hybond-N nylon membrane(Amersham harmacia Biotech, Piscataway, N.J.), cross-linked and stainedwith methylene blue. The filter was then prehybridized with Church'sBuffer (250 mM Na₂HPO₄, 70 mM H₃PO₄, 1 mM EDTA, 1% SDS, 1% BSA in pH7.2) at 65° C. for 1 hour. P501S DNA was labeled with 32P using HighPrime random-primed DNA labeling kit (Boehringer Mannheim).Unincorporated label was removed using MicroSpin S300-HR columns(Amersham Pharmacia Biotech). The RNA filter was then hybridized withfresh Church's Buffer containing labeled cDNA overnight, washed with1×SCP (0.1 M NaCl, 0.03 M Na₂HPO₄.7H₂O, 0.001 M Na₂EDTA), 1% sarkosyl(n-lauroylsarcosine) and exposed to X-ray film.

Using both FACS and Northern analysis, P501S message and protein levelswere found in increase in response to androgen treatment.

EXAMPLE 20 Preparation of Fusion Proteins of Prostate-specific Antigens

The example describes the preparation of a fusion protein of theprostate-specific antigen P703P and a truncated form of the knownprostate antigen PSA. The truncated form of PSA has a 21 amino aciddeletion around the active serine site. The expression construct for thefusion protein also has a restriction site at 3′ end, immediately priorto the termination codon, to aid in adding cDNA for additional antigens.

The full-length cDNA for PSA was obtained by RT-PCR from a pool of RNAfrom human prostate tumor tissues using the primers of SEQ ID NO: 607and 608, and cloned in the vector pCR-Blunt II-TOPO. The resulting cDNAwas employed as a template to make two different fragments of PSA by PCRwith two sets of primers (SEQ ID NO: 609 and 610; and SEQ ID NO: 611 and612). The PCR products having the expected size were used as templatesto make truncated forms of PSA by PCR with the primers of SEQ ID NO: 611and 613, which generated PSA (delta 208-218 in amino acids). The cDNAfor the mature form of P703P with a 6×histidine tag at the 5′ end, wasprepared by PCR with P703P and the primers of SEQ ID NO: 614 and 615.The cDNA for the fusion of P703P with the truncated form of PSA(referred to as FOPP) was then obtained by PCR using the modified P703PcDNA and the truncated form of PSA cDNA as templates and the primers ofSEQ ID NO: 614 and 615. The FOPP cDNA was cloned into the NdeI site andXhoI site of the expression vector pCRX1, and confirmed by DNAsequencing. The determined cDNA sequence for the fusion construct FOPPis provided in SEQ ID NO: 616, with the amino acid sequence beingprovided in SEQ ID NO: 617. The fusion FOPP was expressed as a singlerecombinant protein in E. coli as follows. The expression plasmid pCRX1FOPP was transformed into the E. coli strain BL21-CodonPlus RIL. Thetransformant was shown to express FOPP protein upon induction with 1 mMIPTG. The culture of the corresponding expression clone was inoculatedinto 25 ml LB broth containing 50 μg/ml kanamycin and 34 μg/mlchloramphenicol, grown at 37° C. to OD600 of about 1, and stored at 4°C. overnight. The culture was diluted into 1 liter of TB LB containing50 μg/ml kanamycin and 34 μg/ml chloramphenicol, and grown at 37° C. toOD600 of 0.4. IPTG was added to a final concentration of 1 mM, and theculture was incubated at 30° C. for 3 hours. The cells were pelleted bycentrifugation at 5,000 RPM for 8 min. To purify the protein, the cellpellet was suspended in 25 ml of 10 mM Tris-Cl pH 8.0, 2mM PMSF,complete protease inhibitor and 15 μg lysozyme. The cells were lysed at4° C. for 30 minutes, sonicated several times and the lysate centrifugedfor 30 minutes at 10,000 ×g. The precipitate, which contained theinclusion body, was washed twice with 10 mM Tris-Cl pH 8.0 and 1% CHAPS.The inclusion body was dissolved in 40 ml of 10 mM Tris-Cl pH 8.0, 100mM sodium phosphate and 8 M urea. The solution was bound to 8 ml Ni-NTA(Qiagen) for one hour at room temperature. The mixture was poured into a25 ml column and washed with 50 ml of 10 mM Tris-Cl pH 6.3, 100 mMsodium hosphate, 0.5% DOC and 8M urea. The bound protein was eluted with350 mM midazole, 10 mM Tris-Cl pH 8.0, 100 mM sodium phosphate and 8 Murea. The fractions ontaining FOPP proteins were combined and dialyzedextensively against 10 mM Tris-Cl H 4.6, aliquoted and stored at −70° C.

EXAMPLE 21 Real-Time PCR Characterization of the Prostate-specificAntigen P501S in Peripheral Blood of Prostate Cancer Patients

Circulating epithelial cells were isolated from fresh blood of normalindividuals and metastatic prostate cancer patients, mRNA isolated andcDNA prepared using real-time PCR procedures. Real-time PCR wasperformed with the Taqman procedure using both gene specific primers andprobes to determine the levels of gene expression.

Epithelial cells were enriched from blood samples using animmunomagnetic bead separation method (Dynal A.S., Oslo, Norway).Isolated cells were lysed and the magnetic beads removed. The lysate wasthen processed for poly A+mRNA isolation using magnetic beads coatedwith Oligo(dT)25. After washing the beads in buffer, bead/poly A+ RNAsamples were suspended in 10 mM Tris HCl pH 8.0 and subjected toreversed transcription. The resulting cDNA was subjected to real-timePCR using gene specific primers. Beta-actin content was also determinedand used for normalization. Samples with P501S copies greater than themean of the normal samples+3 standard deviations were consideredpositive. Real time PCR on blood samples was performed using the Taqman™procedure but extending to 50 cycles using forward and reverse primersand probes specific for P501S. Of the eight samples tested, 6 werepositive for P501S and β-actin signal. The remaining 2 samples had nodetectable β-actin or P501S. No P501S signal was observed in the fournormal blood samples tested.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposesof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention. Accordingly, the presentinvention is not limited except as by the appended claims.

SEQUENCE LISTING The patent contains a lengthy “Sequence Listing”section. A copy of the “Sequence Listing” is available in electronicform from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=06620922B1). An electroniccopy of the “Sequence Listing” will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed:
 1. An isolated polynucleotide comprising SEQ ID NO:110, or a fragment thereof comprising at least 50 consecutive nucleotideresidues of SEQ ID NO:
 110. 2. An isolated polynucleotide comprising SEQID NO: 110, or a fragment thereof comprising at least 100 consecutivenucleotide residues of SEQ ID NO:
 110. 3. An isolated polynucleotidecomprising SEQ ID NO: 110, or a fragment thereof comprising at least 500consecutive nucleotide residues of SEQ ID NO:
 110. 4. An isolatedpolynucleotide comprising a sequence having at least 90% identity to theentirety of SEQ ID NO. 110, wherein said polynucleotide is capable ofdetecting expression of SEQ ID NO: 110 in a blood sample, saidexpression being diagnostic of prostate cancer.
 5. An isolatedpolynucleotide comprising a sequence having at least 95% identity to theentirety of SEQ ID NO: 110, wherein said polynucleotide is capable ofdetecting expression of SEQ ID NO: 110 in a blood sample, saidexpression being diagnostic of prostate cancer.
 6. An isolatedpolynucleotide comprising a sequence having at least 99% identity to theentirety of SEQ ID NO: 110, wherein said polynucleotide is capable ofdetecting expression of SEQ ID NO: 110 in a blood sample, saidexpression being diagnostic of prostate cancer.
 7. An isolatedpolynucleotide having at least 95% identity to a sequence consisting ofat least 100 consecutive nucleotide residues of SEQ ID NO: 110, whereinsaid polynucleotide is capable of detecting expression of SEQ ID NO: 110in a blood sample, said expression being diagnostic of prostate cancer.8. An isolated polynucleotide having at least 99% identity to a sequenceconsisting of at least 100 consecutive nucleotide residues of SEQ ID NO:110, wherein said polynucleotide is capable of detecting expression ofSEQ ID NO: 110 in a blood sample, said expression being diagnostic ofprostate cancer.
 9. An isolated polynucleotide consisting of a completecomplement of a polynucleotide according to any one of claims 1-8.
 10. Adiagnostic kit consisting of a polynucleotide according to any one ofclaims 1-9.